CA2231045A1 - Method for producing phage display vectors - Google Patents
Method for producing phage display vectors Download PDFInfo
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- CA2231045A1 CA2231045A1 CA002231045A CA2231045A CA2231045A1 CA 2231045 A1 CA2231045 A1 CA 2231045A1 CA 002231045 A CA002231045 A CA 002231045A CA 2231045 A CA2231045 A CA 2231045A CA 2231045 A1 CA2231045 A1 CA 2231045A1
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
Abstract
The present invention consists in a method for producing a phage display vector which includes causing or allowing recombination between (i) a first vector which includes a sequence encoding a first polypeptide chain of a specific binding pair member; and (ii) a second phage vector which includes a sequence encoding cre-recombinase operatively linked to a control sequence which allows expression of the Cre recombinase and a sequence encoding a second polypeptide chain of a specific binding pair member; wherein the recombination event gives rise to a recombinant phage vector in which expression of the Cre recombinase is substantially inhibited. The present invention also consists in a phage vector which includes a nucleic acid sequence encoding a promoter sequence, a first recombination site downstream and adjacent to the promoter sequence and an open reading frame for cre-recombinase positioned downstream and adjacent to the recombination site such that the promoter drives expression of the Cre recombinase.
Description
, CA 0223l04~ l998-03-03 .:,, t . _ ~
METHOD FOR PRODUCING PHAGE D~:SPLAY VECTORS
The present invention relates to phage display vectors and to methods for producing recombinant phage display vectors.
Functional antigen-binding domains of antibodies (heavy and light chain variable domains) can be displayed on the surface of filamentous bacteriophage. International patent application WO 93/1917Z describes methods, recombinant host cells and kits for production of antibodies displayed on the surface of phage. To produce a ].ibrary of great diversity, recombination occurs between two vectors comprising nucleic acid encoding imrmunoglobulin light and heavy chains respectively producing a recombinant vector encoding the two polypeptide chains. Antibodies displaying the desired antigen binding specificity can be selected from the large number of clones produced by a process called p~nning.
The size of antibody libraries generated using most phage display sysl:ems is limited by the low transformation efficlency of Escherichia coli.
As the size of the m~mmRli~n antibody repertoire is estimated to be in the order of 106 to 108, methods for the generation of larger libraries are required.
A number of groups have investigated the possibility of increasing the size of antibody libraries by combinatorial infection. In principle, heavy and light chains within the initial library or from the original single chain libraries have been systematically shuffled to obtain libraries of exceptionallylarger numbers. Two different mechanisms of site-specific recombination have been used to achieve the association of the two libraries:
1. The lox-Cre system of bacteriophage P1 (~aterhouse et al., 1993;
Griffiths et al., 1994; WO 93/19172) In essence, E.coli is transformed with a repertoire of heavy chain antibody genes (encoded on a plasmid) and then infected with a repertoire of light chain antibody genes (encoded on a phagemid). When Cre recombinase is provided in vivo by infecting the E.coli with P1, the heavy chains residing on the vector and phagemid are exchanged via the lox-P sites. Chain exchange is, however, reversible. Other disadvantages of using this system include a dependency on infection with a Cre-encoding phage and lack of selection for recombination.
METHOD FOR PRODUCING PHAGE D~:SPLAY VECTORS
The present invention relates to phage display vectors and to methods for producing recombinant phage display vectors.
Functional antigen-binding domains of antibodies (heavy and light chain variable domains) can be displayed on the surface of filamentous bacteriophage. International patent application WO 93/1917Z describes methods, recombinant host cells and kits for production of antibodies displayed on the surface of phage. To produce a ].ibrary of great diversity, recombination occurs between two vectors comprising nucleic acid encoding imrmunoglobulin light and heavy chains respectively producing a recombinant vector encoding the two polypeptide chains. Antibodies displaying the desired antigen binding specificity can be selected from the large number of clones produced by a process called p~nning.
The size of antibody libraries generated using most phage display sysl:ems is limited by the low transformation efficlency of Escherichia coli.
As the size of the m~mmRli~n antibody repertoire is estimated to be in the order of 106 to 108, methods for the generation of larger libraries are required.
A number of groups have investigated the possibility of increasing the size of antibody libraries by combinatorial infection. In principle, heavy and light chains within the initial library or from the original single chain libraries have been systematically shuffled to obtain libraries of exceptionallylarger numbers. Two different mechanisms of site-specific recombination have been used to achieve the association of the two libraries:
1. The lox-Cre system of bacteriophage P1 (~aterhouse et al., 1993;
Griffiths et al., 1994; WO 93/19172) In essence, E.coli is transformed with a repertoire of heavy chain antibody genes (encoded on a plasmid) and then infected with a repertoire of light chain antibody genes (encoded on a phagemid). When Cre recombinase is provided in vivo by infecting the E.coli with P1, the heavy chains residing on the vector and phagemid are exchanged via the lox-P sites. Chain exchange is, however, reversible. Other disadvantages of using this system include a dependency on infection with a Cre-encoding phage and lack of selection for recombination.
2. The att recombination system of bacteriophage lambda (Geoffroy et al., 1994) M~ ED ~3H~ET
~P,~AI~
CA 0223104~ 1998-03-03 This process makes use of lambda phage att recombination sites and the Int recombinase to irreversibly create a chimera between plasmid and phagemid vectors carrying respectively, variable light and variable heavy sequences. E.coli is transformed with a ~ lLuire of light chain antibody 5 sequences (encoded on a phagemid). Selection of the recombinant phagemid is possible by the assembly of a chloramphenicol resistance marker upon the correct recombinational event. These features represent certain advRnt~ges over the lox-Cre system, huw~v~r, contrary to the authors' finrling~, vectors possessing two functional E.coli origins of replic~tion are inherently 10 unstable. The fact the att recombination system results in the creation of a potentially hi~shly unstable recombinant, detracts from using it to generate large antibody libraries.
A major problem of the methods used to date is that the recombination process is reversible, thertsroLe stable recombinants are not 15 produced. Furthermore, the methods are dependent on infection with a Cre-expressing bacteriophage and the recombinant phagemid only contains one E.coli replicative origin. Furthermore, the methods used to date do not allow the easy selection of recombinants.
Accordingly, in a first aspect the present invention consists in a 20 method for producing a phage display vector, which method includes causing or allowing recombination b elw~ell (i) a first vector which includes a sequence encoding a first polypeptide chain of a specific binding pair member; and (ii) a second phage vector which includes a sequence encoding Cre 25 recombinase operatively linked to a control sequence which allows expression of the cre recombinase and a sequence encoding a second polypeptide chain of a specific binding pair member;
wherein either the first or second polypeptide chain is fused to a component of a replicable genetic display package which thereby displays 30 the fused polypeptide at the surface of replicable genetic display p~k~ges;
and wherein the recombination event gives rise to a reco~binant phage vector which includes sequences encoding both the first and second polypeptide chains and in which expression of the Cre recombinase is 35 subst~nti~lly inhibited.
W O 97/09436 PCT/AU96~ ''5 By "operatively linked" we mean that a nucleic acid sequence is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is o~eldliv~ly link~d to a coding sequence if it affects the transcription of the sequence.
In a preferred embodiment of the first aspecft of the invention, the second phage vector includes a nucleic acid sequence encoding a ~iol~ r sequence, a first recombination site downstream and adjacent to the promoter sequence and an open reading frame for cre-recombinase positioned downstream and adjacent to the recombi]~ation site, such that the promoter allows expression of the Cre recombinase <;equence, and wherein recombination takes place at the first recombination site such that the open reading frame for Cre recombinase is separated from the promoter sequence.
In a further ~ Ç~ d embodiment the promoter sequence is derived from a promoter for a gene encoding a selectable m~rk~r. Preferably, the selectable mRrk~r is a gene encoding resistance to an Rntimi~:robial agent.
Preferably, the Rntimi~:robial agent is chlor~mph~ ol. In a further embodiment, the promoter is an inducible promoter. The inducible promoter may provide increased control over initialtion of the recombination event.
In a further ~l~Ç~ d embodiment the second phage vector also includes a termin~tnr sequence positioned downstream of the Cre recomlbinase open reading frame in order to prevent undesired transcription of genes downstream of the Cre recombinase gene. The t~rmin~tnr lmay be the rrnLbTlT2 termin~tnr sequence.
In a further preferred embodiment of the invention, the first vector includes a first recombination site upstream and adjacent to a sequence encoding an open reading frame of a selectable mar~:~r, wherein recombination results in the positioning of the sequence encoding the open reading frame of a selectable mArk~r on the recombinant phage vector adjacent to ànd downstream of the promoter sequence such that the ~rolllol~r sequence allows expression of the selectable m~rk~r~ The selectable marker may, for example, be a gene encoding resistance to an antimicrobial gene, or a gene that reacts with a chromogenic compound such as the lacZ gene or the X-gal gene. In a ~l~r~ d embodiment the selectable marker is a gene encoding resistance to an antimicrobial agent. Preferably, the ~ntimi~robia agent is chloramphenicol.
CA 0223104~ 1998-03-03 In a further preferred embodiment the first and second vectors each include a first recombination site and a second recombination site different from the first, site-specific recombination taking place between first recombination sites on the first and second vectors and between second 5 recombination sites on the first and second vectors, but not between the firstrecombination site and the second recombination site on the same vector.
Preferably, the first recombination site is the loxP sequence. The second recombination site is preferably a mutant loxP sequence, more preferably the loxP511 sequence.
In a ~lere~ d form of the invention the first vector is a plasmid and recombination takes place in a bacterial host.
The recombination event may give rise to a second recombinant vector which contains a sequence comprising the Cre recombinase open reading frame. In order to prevent expression of the Cre recombinase from this vector, a terminator sequence may be included upstream of the recombination site of the first vector used in the method of the present invention. This t~rmin~tor sequence should prevent any undesirable expression of the Cre recombinase following the recombination event. This terminator sequence is preferably different to the termin~tnr sequence, if present, on the original second phage vector. The termin~tnr sequence may, for example, be the ~ terminator.
Alternatively, in order to avoid exposure of the recombinant phage vector to Cre recombinase which may be expressed from this second recombinant vector, the recombinant phage particles may be isolated from the host orE~ni.~m In a further ~l~r~ d embodiment of the invention, either the first or second vector further includes (a) a sequence encoding a detectable label;
(b) an inducible stop codon; and (c) a sequence encoding an enzymatic cleavage site such that the sequences described in (a) to (c) are included in the recombinant phage vector. Preferably, the detectable label is the c-myc peptide label, the inducible stop codon is the amber codon and the enzymatic cleavage site is the subtilisin cleavage site.
It will be appreciated that the detectable label allows immllnnlogical detection of expressed antibodies without the need to use antibodies agairlst W O 97/09436 PCT/AU96/00~55 the specific binding pair member. The inducible stop codon allows expression of the polypeptide chains as either a displayed fragment or as a ~ soluble product by using either suppressor or nonsuplressor strains of E.coli for expression. The enzymatic cleavage site facilitates release of the phage from the specific binding pair member.
In a further preferred embodiment, the first vector is pUX-1 and the second phage vector is pMOX-1.
In a second aspect the present invention consists in a phage vector which includes a nucleic acid sequence encoding a promoter sequence, a first recombination site downstream and adjacent to the promoter sequence and an open reading frame for Cre recombinase positioned downstream and adjacent to the recombination site such that the promoter drives expression of the Cre recombinase.
In a ~e~ell~d embodiment of the second aspect of the presenLt invention, the promoter sequence is derived from a promoter for a gene encoding a selectable marker. Preferably, the selectable marker is a gene encoding resistance to an antimicrobial agent. More preferably, the antimicrobial agent is chloramphenicol.
In a further preferred emlbodiment of the second aspect of the present invention the recombination site is the loxP sequence. Preferably, the vector further includes a second recombination site which is different to the first recombination site. The second recombination site is ~l~rel~bly a mutant of the loxP sequence, more preferably the loxP5 11 sequence.
It will be understood that the method of the present invention may be used to produce phage display vectors which are suitable for preparing combinatnri~l libraries of antibodies. The specific binding pair member nnay therefore be an antibody or an antibody fragment. The first polypeptide chain may, for example, be an immunoglobulin heavy chain and the second poly~eyLide chain may be an immllnnglobulin light chain, or vice versa.
The phage vector used in the method of the preserlt invention is designed to transiently express the Cre recombinase until exchange between the two vectors occurs. In particular, the promoter is used to drive Cre recombinase gene expression until recombination occurs. The recombination event subst~nti~lly silences t~ s~ion of the Cre recombinase and therefore gives rise to a stable recombinant phage vector. In a preferred form of the invention the recombination event results in an exchange of -CA 0223104~ 1998-03-03 W O 97/09436 PCT/AU~6 genetic material between the first and second vectors such that the Cre recombinase open reading frame is transferred from the second phage vector to the first vector.
In a preferred form of the invention, the recombinant phage vector not only includes sequences encoding two separate polypeptide chains of a specific binding pair member, but also includes a functional selectable marker. This provides a means for selecting microorganisms cont~ining recombinant phage vectors.
In order that the present invention may be more clearly understood, a preferred form thereof will be described with reference to the following examples and figures in which:
Figure 1. Diagnositic RE analysis of MC05. Lanes 1 to 4 are undigested MC05 clones 1 and Z, MC01 and MC03 respectively. Lanes 5 to 8 are SacI
digested MC05 clones 1 and 2, MC01 and MC03 respectively and in~ic~t~s that MC05 remains undigested whereas MC01 and MC03 are lin~ri.~ed.
Lanes 10 to 13 are XbaI digested MC05 clones 1 and 2, MC01 and MC03 respectively and indicates that MC05 reIn~in.~ llnrligested whereas MC01 and MC03 are line~ri.sed. Lanes 14 to 17 are EcoRI digested MC05 clones 1 and 2, MC01 and MC03 re~e~;liv~ly and in~lic~t~.s that all plasmids are linearised with this RE. Lane 9 is lambda DNA digested with T-Tin~lnT and EcoRI as molecular weight m~rk~r.~.
Figure 2. PCR amplification of the LacVH cassette from MC05. Lanes 2 to 5 are the PCR products of MC05 (lanes 2 and 3) and MC03 (lane 4) and the no template control (lane 5) using MC19 and MC28 as primers. MC05 produces a product of approximately 950bp and MC03 produces a product of 2.5kb as expected. Lane 1 is lambda DNA digested with Hindm and EcoRI as molecular weight markers.
Figure 3. Diagnostic RE analysis of pBIISK-Pcat. Lanes 1 to 9 are mini prep clones 3, 4, 6 to 12 of pBIISK-Pcat digested with BglII and SalI. C is pBIISK(BglII) digested with BglII and SalI. The banding pattern indicates that all clones except clone 11 has the 250bp Pcat fragment cloned into pBIISK(BglII). M is lambda DNA digested with PTin(lTTT and EcoRI as W O 97/09436 PCT/AU9~.'0055S
molecular weight m~rk~rs. Msis pBluescript plasrIL~d DNA digested with Haem as molecular weight markers.
Figure 4. PCR amplification of the Cre recombinase gene. Lanes 1 and 2 is the PCR products of p35Scre using primers MC25 and MC26. p35Scre produces a PCR product of ~llcb as expected. M is lambda DNA digested with ~inrlm and EcoRI as molecular weight markers.
Figure 5. Diagnostic RE analysis of clones of pBIIShcre. C is the control DNA [pBIISK(BglII)~ digested with BssHII and XbaI. Lanes 3, 4, 5, a~d 7 are the respective clones of pBIISKcre digested with Bss]ElII and XbaI as indicated. An XbaI digest was expected to lin~ri~e f;he control and clo~es.
The expected fragment sizes for the pRss~T RE digext were 2.8kb, 861bp and 352bp ~or the clones and 2.9kb and 51bp for the control DNA. The b~nrling pattern indioates that the Cre PCR product has been cloned into pBIISK(B~glII). M is lambda DNA digested with T~in-lTTT and EcoRI as molecular weight markers.
Figure 6. Diagnostic RE analysis of clones of pBIISKterm. The reacffon products of clones 1 to 5 are shown on the left panel and clones 6 to 10 in the right panel. Three diagnostic reactions were ~elrul,llled for each clone: a) digested with SalI and XbaI (left lane); b) PstI (middle lane); and c) undigested (right lane). The XbaI and SalI double digest releases the 380bp Term fragment as indicated. M is lambda DNA digested with ~inflTTT and EcoRI as molecular weight markers. (Note that undigest DNA for clones 5 and 10 are not shown).
Figure 7. Diagnostic RE analysis of clones of pBl[SK TLVH. The control DNA [pBIISK(BglII) - lanes 1 and 2)] and each clone (1, 3, 6, and 9 - lanes 3 to3Q 8) were digested with SacI (left) and PstI, SalI plus BglII (right lane). Asexpected the SacI digest line~ri.ces the plR.crni-l and the triple digest releases the 950bp LacVH fragment and the 380bp Term fragment as indicated - these fragments are not present in the control DNA. M is la:mbda DNA digested with Hindm and EcoRI as molecular weight markers. Ms is pBluescript plasmid DNA digestedL with HaeIII as molecular weigh.t markers.
CA 0223104~ 1998-03-03 Figure 8. Diagnostic RE analysis of pMOX. Clones 9 and 11 of pMOX were digested with XbaI and ~inrlm (lanes 1 and 2 respectively) which releases the 250bp Pcat fragment that is the same size as an XbaI and ~inrlm digest of pBIISKPcat-Cre (lane 3). Similarly, clones 9 and 11 of pMOX were digested with SalI and PstI (lanes 4 and 5 respectively) which releases the 380bp Term fragment that is the same size as a SalI and PstI digest of pBIISK-TLVH (lane 6). M is lambda DNA digested with ~in~lTTT and EcoRI as molecular weight markers. Ms is pBluescript plasmid DNA digested with Haem as molecular weight markers.
Figure 9. Diagnostic screening for pUX. Twelve clones were digested with PstI (left lane) and undigested (right lane) to screen for the inclusion of the LacVH fragment cloned into pUTcat. Lane C is similarly digested vector pUlcat. Clone 11 clearly has the lacVH fragment cloned into the PstI site since it is releasing a fragment of ~950bp that is a simil~r size to the LacVH
PCR product (lane L) and which the control DNA does not have (left lane C).
It is also clearly evident that the llntligR.sted supercoiled DNA is larger thanthe control DNA (right lane C). M is lambda DNA digested with ~inrlm and EcoRI as molecular weight markers.
Figure 10. Analysis of the orientaffon of the LacVH insert of pUX clone 11.
pUX clones 10, 11 and 12 were digested with BalI (lanes 1, 2 and 3 respectively). A 700bp fragment is released for the correct orientation and 1120bp for the incorrect orientation. Lane 2 clearly intlir:~tRs that the LacVH
fragment in clone 11 is in the correct orientation. pUX clones 10, 11 and 12 were also digested with BglII (lanes 5, 6 and 7 respectively) and in~licRtRs that pUX is linR~ri.sed as expected. M is lambda DNA digested with ~in~m and EcoRI as molecular weight m~rkRrs.
Figure 11. Diagnostic RE analysis of pUX-TT. The isolate clone of pUX-TT
was digested with XhoI (lane 1), SpeI (lane 2), XhoI and SpeI (lane 3) and undigested (lane 5). The double digest releases the 600bp TT heavy chain DNA sequence. Lane 4 is XhoI and SpeI digested pUX. M is lambda DNA
digested with ~in(lm and EcoRI as molecular weight markers.
WO ~7/09436 PCT/AU96/00555 Figure 12. Diagnostic RE analysis of pMOX-TT. T~Telve clones (1 to 12) were digested with SacI and XbaI (left lane) and uncligested (right lane) to screen for the inclusion of the TT light chain DNA firagment cloned into pMOX. Lane C is similarly digested vector pMOX. All clones clearly have 5 the TI fragment cloned into the SacI and XbaI sites since a fragment of ~600bp is released which the control DNA does not have (left lane C). It is also clearly evident that the undigested supercoiled DNA for each clone is larger than the control DNA (right lane C). M is lambda DNA digested with ~inclTTT and EcoRI as molecular weight m~rk~rs.
Figure 13. Diagramatic representations of pMOX (a) and pUX (b).
Figure 14. Diagramatic representation of the propo~ed me-:h~ni.~rn of in vivo recombination between the pMOX and pUX vectors.
Figure 15. Diagramatic representation of the recombinant phage vector pMUX.
EXAMP~.ES
20 Construction of the Lac VH cassette The LacVH cassette was based on the antibocly expression vector MCO1 (Ward et al., 1996), which contains cloning sites for both the heavy and light chain DNA sequences. The first part of the construction of the cassette was to remove the XbaI restriction endonuclease (RE) site (3' cloning 25 site of the light chain) from MCO1. Following digestion with XbaI, MCO1 was treated with klenow fragment of DNA polymerase I (blunted) and subsequently religated and transformed into E. coli strain XLl-blue. Twenty transformants were selected and subjected to RE and gel electrophoretic analysis. Fifteen of these clones were found to be devoid of the XbaI site.
30 One construct, designated MCO4, was retained for further work. The second part of the construction was to remove the light chain leader region from EcoRI to SacI sites. MCO4 was digested with SacI, gel purified and redigested with EcoRI. The double cut MCO4 DNA was then subjected to klenow treatment to 'fill-in' the cohesive ends, ligatecl and transformed i~to 35 XLl-blue. This construction procedure desllvy~d the SacI site but retains theintegrity of the EcoRI site. Twenty transformants were randomly selected CA 0223104~ 1998-03-03 and subjected to RE analysis with EcoRI, SacI and XbaI. At least 10 clones were isolated that displayed the expected profile. This construct is referred toas MC05.
In order to positively determine whether these recombinants were 5 devoid of the 86bp light chain leader sequence, the nucleotide sequence of this region was determined for four clones using MC03.for (MC28) PCR
primer. All four clones were found to be devoid of the said sequence and one clone was retained. Figure 1 indicates that MC05 but not MC01 and MC03 ~which has stuffer fragments cloned into the light and heavy chain cloning 10 sites) are linearized by SacI and XbaI, while all three MCO v~:~Lols are linearized with EcoRI. As expected, it is also evident that MC05 is slightly smaller than MC01.
A 956bp fragment corresponding to position 90 and 987 of MC05 was then amplified by the polymerase chain reaction (PCR) using primers MC19 15 and MC28. The resultant PCR product, designated as the LacVH cassette, contained starting from the 5' end, a unique Pstl site, the lacZ promoter and operator sequence (lacZ P/O), the pel B leader sequence, XhoI and SpeI
unique RE sites, a myc tag, an amber point mutation, a subtilisin open reading frame (ORF), the gene m ORF, a translational stop codon, a mutated 20 loxP site (designated loxP5ll) and a unique BglII site [Figure 2].
The loxp5ll site was introduced into the cassette with the reverse primer MC19, which incorporated a loxP5ll DNA sequence. Intramolecular excision events have been noted where recombination occurs between two loxP sites that are in the same orientation of the DNA substrate (Abremski et 25 al., 1986). In order to avoid this problem in the ffnal construct (pMOX), andin the recombinatorial vector, the loxP511 sequence was positioned in the opposite orientation with repect to the wild type loxP site.
The ~mrlified LacVH cassette was subsequently used in the construction of both the acce~Lor phage vector and the donor plasmid vector.
Construction of pMOX
This vector was based on MC01 and was designed to transiently express 'cre' recombinase until an exr~h~nge between v~cLul~ has occured.
The chloramphenicol acteyltransferase ~CAT) gene promoter (Pcat) is used to 35 drive the cre-recombinase gene t,,~ ssion. Between the Pcat promoter and W O 97/09436 PCT/AU~ 5 the Cre gene is the loxP sequence which is one of tvw designated recombination sites.
Construction of pMOX involved the PCR am~plificatior~ of four discrete fragments that were cloned into MCO1. These fragments are ~ 5 referred to as: Pcat, which contains the chloramphenicol promoter and theloxP site 3' of the promoter; Cre, which contains the sequences necessary to express the creatin recombinase protein; Term, which contains ribonucIease terminator sequences; and LacVH, which contains the cloning site for the heavy chain of an antibody and the loxP511 site 3' of the gene m stop codon.
Construction of the Pcat-Cre cassette The Pcat element was successfully amplifiedL from the CAT gene residing on the plasmid pACYC184 using the primers MC27 and MC17. To determine the efficacy of the amplified Pcat element and whether the loxP
site interfered with transcription, the PCR product was cloned into the promoter-probed vector pklc232-8. Following blunting with Klenow to remove the terminal A's, the Pcat PCR product was dLigested with ~inrlTTT and cloned into the T~in(lmlsmaI site of pkk232-8. The ligation mix was used to transformed XL1-blue cells and promoter efficiency det~rmin~d by spreading the cells on LB agar plates contRining varying (10-50mg) concentrations of chloramphenicol. Five Pcat clones were isolated andL were subjected to PCR
and gel electrophoretic analysis. All five were found to harbour the Pcat element (including the loxP site) indicating that the ]?cat promoter was not compromised because of the presence of the loxP site.
Attempts to clone the Pcat element into the vector pBltSK(BglII) [kindly provided by Dr. Guy Lyons, K~n~m~tsu Laboratory, Universi~ of Sydney] using the unique XbaI and T-TinrlTTT RE sites ~rere unsuccessful. To facilitate cloning of the Pcat element, two further prirn~r.~ were designed (MC50 and MC51) that amplified the Pcat PCR product and also contained additional Hindm RE sites at both ends of the PCR product. The Pcat PCR
fragment and pBltSK~BglII) were digested with T~in~lTrT RE, puri:~ied in an a~garose gel, ligated, and used to transform XL1-blue cells. Twelve transformants were selected and subjected to RE and gel electrophoretic analysis. Eight clones were foumd to contain the Pcat PCR fr~gm~nt and one clone was selected for further work and named pBltS]E~-Pcat. Figure 3 CA 0223104~ 1998-03-03 indicates that the Pcat fragment is released by ~in~lm RE digestion of this plasmid.
The cre recombinase ORF (including the ribosome binding site RBS) was amplified from pBS157 ~kindly provided by Dr Peter Waterhouse, CSIRO
5 Division of Plant Industry, Canberra, Australia] with primers that were designed using the E. coli P1 nucleotide sequence derived by Sternberg et al.
(1986). These two primers (MC25 and MCZ6) incorporated unique ~inrlm and SalI RE sites. After successful ~mplific~tion of the Cre fragment (Figure 4) the product was digested with ~in~lm and SalI, and ligated into ~in~lm 10 and SalI digested pBltSK(BglII). The ligation mix was used to transform XL1-blue cells and eleven of 12 single colonies screened by RE analysis were shown to contain the Cre gene. Four clones further characterised by RE
analysis showed ~he expected b~n-ling patterns (Figure 5). Clone 3 was designated pBltSK-Cre and used for further manipulations.
The Pcat-Cre cassette was constructed by cloning the Pcat fragment (from pBltSK-Pcat) into pBltSK-Cre. This was ~elfolllled by digesting both pBltSK-Pcat and pBltSK-Cre with XbaI and Hin-lm The 4kb fragment from pBltSK-Cre and the 250bp fragment from pBltSK-Pcat were agarose gel purified and ligated The ligation products were used to transform XLl-blue cells and 1Z single colonies were isolated. Initial screening indicated that 10 of the 12 clones had the Pcat fragment cloned into the pBltSK-Cre vector.
Further diagnostic digests were performed on the clones and one was designated pBltSK-PcatCre and used in subsequent manipulation.
Construction of Term-LacVH cassette In order to prevent read through and interference from Pcat, the E.
Coli. 5s ribosomal transcription termin~tor ~rrnbT1T2) [Brosius et al., 19811 was positioned adjacent to the 3' end of the Cre ORF. The rrnbTlT2 t~rmin~tor was successfully amplified from pkk232-8 using the primers MC24 and MC23, generating a product of 340bp. The transcriptional terminator product was restricted with SalI and PstI and ligated into the cloning vector pSP72. The fragment was subsequently extracted from this vector by digestion with SalI and PstI, and ligated into Pstl and SalI digested pBltSK(BglII). The ligation mix was used to transform XL1-blue cells and 12 single colonies were isolated and screened for the inclusion of the Term fragment. All 12 colonies contained the Term fragment (figure 6) and one CA 0223l045 l998-03-03 W O 97/09436 PCT/AU96~ S~' was selected, further characterized and designated pBltSK-Term. This plasmid was used for subsequent manipulations.
The next stage in acceptor phage vector comstruction was to clone the LacVH gene into pBltSK-Term, to produce the Term-LacVH cassette. As - 5 described above a 956bp fragment had been PCR ~mlplified from MC05. New primers (MC48 and MC49) were rlesigned to extend ~he ends of that fragment, adding ~intlm cloning sites to both ends. A fragment of a~ ;ate size was ~mplifi~hle using these two primers and the original PCR product as template. The PCR product and pBllSK-Term were then digested with PstI and BglII, gel purified and ligated. The ligation reaction was used to transform XL1-blue cells and 12 single colonies were isolated and screened for the inclusion of the LacVH fragment. Eleven of the 12 colonies contained the LacVH gene. Further RE rliRgrnostic digests were performed (Figure 7) and one clone was selected andl designated pBltSK-TLVH. This clone was used for subsequent DNA manipulations.
Construction of pMOX
The final step in the construction of pMOX involved digesting: a) MC01 with XbaI and BglII; b) pBltSK-TLVH with B~slII and SalI; and c) pBltSK-PcatCre with SalI and XbaI. The MC01 vect~r, TLVH and PcatCre cassettes were purified in an agarose gel, mixed and ligated. The ligation mix was used to transform XLl-Blue cells and 12 colonies were isolated and screened for the correct construction. Figure 8 inflic~t~s expected restrictio digest patterns of the resultant pMOX vector.
Construction of pUX
Construction of pUX was based on pUC19 (Yanisch et al., 1985).
Since both the donor and acce~lor vectors possessed the same antibiotic resistance (~mpir,illin - ApR) determin~nt, the ApR gene of pUC19 was replaced with a PCR amplified Tetracyclin resistance (TcR). The entire TcR
gene was amplified from pACYC184, using primers that included PvuI RE
sites at both ends of the PCR product (MC21 and MC22). The TcR gene was restricted with PvuI, and ligated with the 179obp Pvu~ restricted fragment of pUC19. This fragment was partially devoid of the ApR coding region (thereby m~king it sensitive to Ap), as well as the cod~ing region for lacZ. Theligation ~ cLul~ was then transformed into the Tc E. coli sensitive strain CA 0223l04~ l998-03-03 W O 97/09436 PCT/AU9~ 'C' NM522, and recombinants were selected for resistance to Tc. Following PvuI
RE analysis one clone (designated pUT-1, 3209bp) was retained.
The reIn~inrle.r of the vector was constructed by cloning two PCR
generated fragments into the MCS of pUC19. These fragments are referred to as: CAT (which contains the sequences that induce resistance to chloramphenicol); and LacVH (which contains the cloning site for the heavy chain of an antibody).
The chlor~mph~ir.ol resistance gene (the ORF excluding its promoter region) was PCR amplified using primers MC18 and MC20. The cat gene primers were based on the nucleotide sequence of the cat gene of the plasmid Tn9 (Alton and Vapnek, 1979). After amplifcation the loxP-cat gene was restriced with SacI/PstI and ligated into SacIlPstI restricted pl~T-1 vector.
The ligation ~ e was transformed into NM5Z2 and mini-plasmid lysate preps of eighteen randomly chosen isolates were analysed by RE and gel electrophoresis. Additional characterization of four clones with PCR
revealed that all four clones (designated pl~Tcat) contained, the loxP-cat gene.
The next step in the construction of pUX was to clone the lacVH gene into the Psff site. This was accomplished by amplifying the LacVH PCR
fragment described earlier with primers (MC48 and MC58) that added a PstI
site to both ends of the LacVH gene. The PCR fragment and pUTcat was restricted with PstI, the DNA was purified in an agarose gel and the DNA
ligated. The ligation mixture was transformed into NM522 cells and mini-plasmid lysate preps of eighteen randomly isolated clones were analysed by RE and gel electrophoresis. One clone contained the DNA insert (Figure 9) which, with additional RE analysis, was shown to be in the correct orientation (Figure 10). This clone was rlesign~ted pUX.
~ vivo recombination of pUX and pMOX
To test for 'in vivo' recombination between pUX and pMOX, the heavy chain of a tetanus toxoid (~) Fab was cloned into pUX, while the light chain was cloned into pMOX. Phage was then prepared from pMOX-TT, and was used to infect log phase HB2151 cells previously transfected with pUX-TI.
Construction of pUX-TT and pMOX-TT
CA 0223l045 l998-03-03 W O 97/09436 PCT/AU~
The tetanus toxoid heavy chain was RE digested with XhoVspeI and purified in an agarose gel from MC01-TT. This frag;ment was ligated into - XhoVspeI RE digested pUX. The ligation llPLcLIlre was used to transform HB2151 cells and a single colony was isolated. This; colony was screened using RE digests and gel electrophoresis for the inclusion of the TT heavy chain DNA. Figure 11 indicates that this clone contained the IT heavy chain DNA sequences. The tetanus toxoid light chain was, RE digested with SacI/XbaI and purified in an agarose gel from MC01-TT. This fragment was ligated into SacVXbaI RE digested pMOX. The ligation ~ re was used to 0 transform XL1-blue cells and 1Z colonies were isolated and screened using RE digests and gel electrophoresis for the inclusion of the TT light chain.
Figure 12 indicates that all 12 colonies contained the TT light chain DNA
sequences. These two plasmids were referred to as pUX-lT and pMOX-TT.
Preparation of pMOX-TT phage.
pMOX-TT was transformed into XL1-blue and 250,u1 of an overnight culture was used to inoculate 2YT cont~ining 2% glucose, carb~nicillin (carb -50~Lg/ml)tetracycline (tet - 1o,ug/ml) and then rescue with helper phage (VCS-M13). The culture was incubated for 2 hours at 37~C with shaking and ZO thencentrifuged at room L~nlyeltlture at 4500rpm for 15 minutes. The bacterial pellet was resuspended in 50ml of 2YT containing carb 50,ug/ml, tetlO~lg/ml and k~n~mycin 70~1g/ml and incubated overnight at 37~C with sh~king. The bacteria were pelleted by centrifugation at 8000rpm at room temperature, The phage were precipitated from the supernatant by ~flrling 1/5vol 20~ PEG6000/2.5M NaCl, mixing and leaving on ice with occasional sh~king for 1 hour. The phage were collected by cenltrifugation at 8000rpm for 20 lminutes at 4~C. The pellet was resuspended in 500,ul of 19~o BSA, and bacterial debris was removed by a high speed centrifugation in a microfuge tube and stored at 4~C.
k2 vivo recombination between pUX-TT and pMOX-lT
A 1ml culture of pUX transformed HB2151 cells in media cont~ining tetracycline (OD600 = 0.8) were infected with lx109 pMOX-~ phage a~d allowedL to stand at 37~C for 30 minutes. Carb (2511g/rnl) was added and the culture incubated for 30 minutes with sh~king. Further carb (25,u~/ml) was added and incubated with .ch~king for a further 3 hours. An ali~uot of cells CA 0223l045 l998-03-03 added and incubated with shaking for a further 3 hours. An aliquot of cells (250,u1) was obtained 1 and 4 hours after infection and plated onto: a) Carb;
b) Tet; c) ChlorlO; d) Chlor50; e) Carb/ChlorlO; and f) Carb/Chlor50.
Since pMOX is tet sensitive and pUX is carb sensitive, growth of cells 5 in culture indicated that the cells contain both pMOX and pUX.
Chloramphenicol further selects for cells that have recombinants contRining the pMUX vector since chlor~mrh~ir.ol resistance is acquired only after recombination. A lawn of colonies were observed on both chlor and carb/chlor plates (at both concentrations of chlor) following infection of 10 pMOX-TT into the culture contRining HB2151/pUX-TT indicating that recombination had occurred.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the 15 invention as broadly described. The present embodiments are, th~le:rule, to be considered in all respects as illustrative and not restrictive.
List of Primers MC03.for (MC28) 5'.. ACC TGC AGA ACA GTG AGC GCA ACG CAA T.. 3' Pstl site for cloning MC03.rev (MC19) 5'..GAA GAT CTT C~AT AAC TTC GTA TAT ACA TAT
GTA TAC GAA GTT ATlG CGG CCG CTT AAA TTA AIT AGC..3' BglII site for cloning and ~loxP511] recombination site 25 Pcat.for (MC50) 5'CCC AAG Cl~ GGG CTC TAG AGC CGA CGC ACT TTG
CGC CGA AT..3' Forward primer for Pcat PCR fragment; XbaI and ~dm PcatPCR.rev (MC5 1) 5 '. .CCC AAG CTI CCC AAG C~T GGG ATA ACT TCG
TAT AGC ATA CAT TAT3' Reverse primer for Pcat PCR fragment with LoxP site; HindIII.
Pcat.for (MC27) 5'..GCT CTA GAG CCG ACG CAG TTT GCG CCG A11..3' Reverse primer for the ~mrlific~tion of LacVH of pMOX.
Pcat.rev (MC17) 5'..GGG AAG C~ CCC ~ATA ACT TCG TAT AGC ATA
CAT TAT ACG AAG TTA TlTC GAT AAC TCA AAA AAT ACG
CC.. 3' Reverse PCR primer for ~mplifir:~tion of the CAT gene lol~r fro Tn9. HindIII for cloning and ~loxPl Cre.rev (MC25) 5'..ACG CGT CGA CCG CGT TAA TGG CTA ATC GC..3' Reverse primer for the amplification of cre recombinase of pMOX.
Cre.for (MC26) 5'..CCC AAG CTT CTG AGT GTT AAA TGT CCA ATT
TAC.. 3' Forward primer for the ~mplific~tic ~ of cre recombinase of pMOX.
CA 0223l045 l998-03-03 WO97/09436 PCT/AU9~ 555 Term.for (MC24) 5'..ACG CGT CGA CCA GAA GTG AAA CGC CGT A..5' Forward primer for the ~mplification of terlrnin~tnr for pMOX.
Term.rev (MC23) 5'..TTC TGC AGT TCC TGA TGA TGC AAA AAC GAG
GC..3' Reverse primer for the ~mplificatiorl of t~rmin~tor of pMOX.
5 MCO5.for (MC48) 5'..CCC AAG c7'r GGG ACC TGC AGA ACA GTG AGC
GCA ACG CAA T. . 3' Forward primer for P('R of LacVH of pMOX;
PslI, ~ndIII
MCO5.rev (MC49) 5'..CCC AAG C7T GGG AAG ATC TTC ATA ACT TCG
TAT ATA CAT A..3' Reverse primer for PClR of LacVH with loxP
site; BglII, HindIII.
tet.rev ~MC21) 5'..TAC GAT CGT ATT CAC AGT TCT CCG CAA GA..3' Reverse primer for the ~mplifir:~tion of tetR for pUX.
tet.for (MC22) 5'..ATC GAT CGA TCA AAT GTA GCA CCT GAA GTC AG..3' Forward primer for the ~mplification of tetR for pUX.
5 cat.for (MC18) 5'..CGA GCI'CGrA TAA CTT CGT A'~A ATG TAT GCT ATA
CGA AGT TATl GAT TTT CCA GGA GCT~AG GAA..3' Forward PCR primer for the ~mplific~tion of the CAlr resistance gene from Tn9; SacI, [lo~Pl cat.rev (MC20) 5'..TIT GCA GAT CGT CAA TTA CCT CCA CG..3' Reverse Z0 primer for RmplifiC~tion of CatR gene for pUX.
MCO.rev PPCR (MC58~ 5'..AAA ACT GCA GCC AAG AllC ITC ATA ACT
TCG TAT ATA CAT A..3' Reverse PCR prinner for LacVH MCO PC~
product; Pstl, BglII
J CA 0223104~ 1998-03-03 ._. ~ - ' ~ -. ~
_ . . ~ ,, BIBLIOGRAPHY
Abremski, K., Frommer, B. and Hoess, R. H. (19~6) T.inking-Ilumber changes in the DNA substrate during Cre-mediated loxP site-specific recombination.
Journal of Molecular Biology 192(1):17-26.
Alton, N. K. and Vapnek, D. (1979) Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9. Naf.ure 28Z(5741):864-9.
Brosius, J., Dull, T. J., Sleeter, D. D. and Noller, E3:. F. (1981) Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. Journal of Molecular Biology 148(2):107-27.
Geoffroy, R., Sodoyer. R, and Aujame, L. (1994) A new phage display system to construct multicombinatorial libraries of very large antibody repertoires.
Gene. 151:109-113.
Griffiths, A. D., Williams, S.C., Hartley, O, Tomlinson, I.M., Waterhouse, P., Crosby, W.L, Kontermann, R,E., Jones, P.T., Low, N.M., Allison, T.J (1994) Isolation of high affinity hurnan antibodies directly from large synthetic repertoires. Embo Journal. 13 :3245-60.
Sternberg, N., Sauer, B., Hoess, R. and Abremski, K. (1986) Bacteriophage P1 cre gene and its regulatory region. Evidence for multiple promoters and for regulation by DNA methylation. Journal of Molecular Biology 187(2): 197-Z 12.
Ward, R. L., Clark, M. A., Lees, J., Hawkins, N. J. (1996) Retrieval of human antibodies from phage-display libraries using enzymatic clevage. J.
Immunol. Methods. 189: 73-82.
Waterhouse, P., Griffiths A.D., Johnson, K.S., and. Winter. G. (1993) Combinatorial infection and in vivo recombination: a strategy for m;~king large phage antibody repertoires. Nucleic Acids Research. 21:2265-6.
Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUClg vectors. Gene 33(1):103-19.
;HEET
~P~/AlJ
~P,~AI~
CA 0223104~ 1998-03-03 This process makes use of lambda phage att recombination sites and the Int recombinase to irreversibly create a chimera between plasmid and phagemid vectors carrying respectively, variable light and variable heavy sequences. E.coli is transformed with a ~ lLuire of light chain antibody 5 sequences (encoded on a phagemid). Selection of the recombinant phagemid is possible by the assembly of a chloramphenicol resistance marker upon the correct recombinational event. These features represent certain advRnt~ges over the lox-Cre system, huw~v~r, contrary to the authors' finrling~, vectors possessing two functional E.coli origins of replic~tion are inherently 10 unstable. The fact the att recombination system results in the creation of a potentially hi~shly unstable recombinant, detracts from using it to generate large antibody libraries.
A major problem of the methods used to date is that the recombination process is reversible, thertsroLe stable recombinants are not 15 produced. Furthermore, the methods are dependent on infection with a Cre-expressing bacteriophage and the recombinant phagemid only contains one E.coli replicative origin. Furthermore, the methods used to date do not allow the easy selection of recombinants.
Accordingly, in a first aspect the present invention consists in a 20 method for producing a phage display vector, which method includes causing or allowing recombination b elw~ell (i) a first vector which includes a sequence encoding a first polypeptide chain of a specific binding pair member; and (ii) a second phage vector which includes a sequence encoding Cre 25 recombinase operatively linked to a control sequence which allows expression of the cre recombinase and a sequence encoding a second polypeptide chain of a specific binding pair member;
wherein either the first or second polypeptide chain is fused to a component of a replicable genetic display package which thereby displays 30 the fused polypeptide at the surface of replicable genetic display p~k~ges;
and wherein the recombination event gives rise to a reco~binant phage vector which includes sequences encoding both the first and second polypeptide chains and in which expression of the Cre recombinase is 35 subst~nti~lly inhibited.
W O 97/09436 PCT/AU96~ ''5 By "operatively linked" we mean that a nucleic acid sequence is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is o~eldliv~ly link~d to a coding sequence if it affects the transcription of the sequence.
In a preferred embodiment of the first aspecft of the invention, the second phage vector includes a nucleic acid sequence encoding a ~iol~ r sequence, a first recombination site downstream and adjacent to the promoter sequence and an open reading frame for cre-recombinase positioned downstream and adjacent to the recombi]~ation site, such that the promoter allows expression of the Cre recombinase <;equence, and wherein recombination takes place at the first recombination site such that the open reading frame for Cre recombinase is separated from the promoter sequence.
In a further ~ Ç~ d embodiment the promoter sequence is derived from a promoter for a gene encoding a selectable m~rk~r. Preferably, the selectable mRrk~r is a gene encoding resistance to an Rntimi~:robial agent.
Preferably, the Rntimi~:robial agent is chlor~mph~ ol. In a further embodiment, the promoter is an inducible promoter. The inducible promoter may provide increased control over initialtion of the recombination event.
In a further ~l~Ç~ d embodiment the second phage vector also includes a termin~tnr sequence positioned downstream of the Cre recomlbinase open reading frame in order to prevent undesired transcription of genes downstream of the Cre recombinase gene. The t~rmin~tnr lmay be the rrnLbTlT2 termin~tnr sequence.
In a further preferred embodiment of the invention, the first vector includes a first recombination site upstream and adjacent to a sequence encoding an open reading frame of a selectable mar~:~r, wherein recombination results in the positioning of the sequence encoding the open reading frame of a selectable mArk~r on the recombinant phage vector adjacent to ànd downstream of the promoter sequence such that the ~rolllol~r sequence allows expression of the selectable m~rk~r~ The selectable marker may, for example, be a gene encoding resistance to an antimicrobial gene, or a gene that reacts with a chromogenic compound such as the lacZ gene or the X-gal gene. In a ~l~r~ d embodiment the selectable marker is a gene encoding resistance to an antimicrobial agent. Preferably, the ~ntimi~robia agent is chloramphenicol.
CA 0223104~ 1998-03-03 In a further preferred embodiment the first and second vectors each include a first recombination site and a second recombination site different from the first, site-specific recombination taking place between first recombination sites on the first and second vectors and between second 5 recombination sites on the first and second vectors, but not between the firstrecombination site and the second recombination site on the same vector.
Preferably, the first recombination site is the loxP sequence. The second recombination site is preferably a mutant loxP sequence, more preferably the loxP511 sequence.
In a ~lere~ d form of the invention the first vector is a plasmid and recombination takes place in a bacterial host.
The recombination event may give rise to a second recombinant vector which contains a sequence comprising the Cre recombinase open reading frame. In order to prevent expression of the Cre recombinase from this vector, a terminator sequence may be included upstream of the recombination site of the first vector used in the method of the present invention. This t~rmin~tor sequence should prevent any undesirable expression of the Cre recombinase following the recombination event. This terminator sequence is preferably different to the termin~tnr sequence, if present, on the original second phage vector. The termin~tnr sequence may, for example, be the ~ terminator.
Alternatively, in order to avoid exposure of the recombinant phage vector to Cre recombinase which may be expressed from this second recombinant vector, the recombinant phage particles may be isolated from the host orE~ni.~m In a further ~l~r~ d embodiment of the invention, either the first or second vector further includes (a) a sequence encoding a detectable label;
(b) an inducible stop codon; and (c) a sequence encoding an enzymatic cleavage site such that the sequences described in (a) to (c) are included in the recombinant phage vector. Preferably, the detectable label is the c-myc peptide label, the inducible stop codon is the amber codon and the enzymatic cleavage site is the subtilisin cleavage site.
It will be appreciated that the detectable label allows immllnnlogical detection of expressed antibodies without the need to use antibodies agairlst W O 97/09436 PCT/AU96/00~55 the specific binding pair member. The inducible stop codon allows expression of the polypeptide chains as either a displayed fragment or as a ~ soluble product by using either suppressor or nonsuplressor strains of E.coli for expression. The enzymatic cleavage site facilitates release of the phage from the specific binding pair member.
In a further preferred embodiment, the first vector is pUX-1 and the second phage vector is pMOX-1.
In a second aspect the present invention consists in a phage vector which includes a nucleic acid sequence encoding a promoter sequence, a first recombination site downstream and adjacent to the promoter sequence and an open reading frame for Cre recombinase positioned downstream and adjacent to the recombination site such that the promoter drives expression of the Cre recombinase.
In a ~e~ell~d embodiment of the second aspect of the presenLt invention, the promoter sequence is derived from a promoter for a gene encoding a selectable marker. Preferably, the selectable marker is a gene encoding resistance to an antimicrobial agent. More preferably, the antimicrobial agent is chloramphenicol.
In a further preferred emlbodiment of the second aspect of the present invention the recombination site is the loxP sequence. Preferably, the vector further includes a second recombination site which is different to the first recombination site. The second recombination site is ~l~rel~bly a mutant of the loxP sequence, more preferably the loxP5 11 sequence.
It will be understood that the method of the present invention may be used to produce phage display vectors which are suitable for preparing combinatnri~l libraries of antibodies. The specific binding pair member nnay therefore be an antibody or an antibody fragment. The first polypeptide chain may, for example, be an immunoglobulin heavy chain and the second poly~eyLide chain may be an immllnnglobulin light chain, or vice versa.
The phage vector used in the method of the preserlt invention is designed to transiently express the Cre recombinase until exchange between the two vectors occurs. In particular, the promoter is used to drive Cre recombinase gene expression until recombination occurs. The recombination event subst~nti~lly silences t~ s~ion of the Cre recombinase and therefore gives rise to a stable recombinant phage vector. In a preferred form of the invention the recombination event results in an exchange of -CA 0223104~ 1998-03-03 W O 97/09436 PCT/AU~6 genetic material between the first and second vectors such that the Cre recombinase open reading frame is transferred from the second phage vector to the first vector.
In a preferred form of the invention, the recombinant phage vector not only includes sequences encoding two separate polypeptide chains of a specific binding pair member, but also includes a functional selectable marker. This provides a means for selecting microorganisms cont~ining recombinant phage vectors.
In order that the present invention may be more clearly understood, a preferred form thereof will be described with reference to the following examples and figures in which:
Figure 1. Diagnositic RE analysis of MC05. Lanes 1 to 4 are undigested MC05 clones 1 and Z, MC01 and MC03 respectively. Lanes 5 to 8 are SacI
digested MC05 clones 1 and 2, MC01 and MC03 respectively and in~ic~t~s that MC05 remains undigested whereas MC01 and MC03 are lin~ri.~ed.
Lanes 10 to 13 are XbaI digested MC05 clones 1 and 2, MC01 and MC03 respectively and indicates that MC05 reIn~in.~ llnrligested whereas MC01 and MC03 are line~ri.sed. Lanes 14 to 17 are EcoRI digested MC05 clones 1 and 2, MC01 and MC03 re~e~;liv~ly and in~lic~t~.s that all plasmids are linearised with this RE. Lane 9 is lambda DNA digested with T-Tin~lnT and EcoRI as molecular weight m~rk~r.~.
Figure 2. PCR amplification of the LacVH cassette from MC05. Lanes 2 to 5 are the PCR products of MC05 (lanes 2 and 3) and MC03 (lane 4) and the no template control (lane 5) using MC19 and MC28 as primers. MC05 produces a product of approximately 950bp and MC03 produces a product of 2.5kb as expected. Lane 1 is lambda DNA digested with Hindm and EcoRI as molecular weight markers.
Figure 3. Diagnostic RE analysis of pBIISK-Pcat. Lanes 1 to 9 are mini prep clones 3, 4, 6 to 12 of pBIISK-Pcat digested with BglII and SalI. C is pBIISK(BglII) digested with BglII and SalI. The banding pattern indicates that all clones except clone 11 has the 250bp Pcat fragment cloned into pBIISK(BglII). M is lambda DNA digested with PTin(lTTT and EcoRI as W O 97/09436 PCT/AU9~.'0055S
molecular weight m~rk~rs. Msis pBluescript plasrIL~d DNA digested with Haem as molecular weight markers.
Figure 4. PCR amplification of the Cre recombinase gene. Lanes 1 and 2 is the PCR products of p35Scre using primers MC25 and MC26. p35Scre produces a PCR product of ~llcb as expected. M is lambda DNA digested with ~inrlm and EcoRI as molecular weight markers.
Figure 5. Diagnostic RE analysis of clones of pBIIShcre. C is the control DNA [pBIISK(BglII)~ digested with BssHII and XbaI. Lanes 3, 4, 5, a~d 7 are the respective clones of pBIISKcre digested with Bss]ElII and XbaI as indicated. An XbaI digest was expected to lin~ri~e f;he control and clo~es.
The expected fragment sizes for the pRss~T RE digext were 2.8kb, 861bp and 352bp ~or the clones and 2.9kb and 51bp for the control DNA. The b~nrling pattern indioates that the Cre PCR product has been cloned into pBIISK(B~glII). M is lambda DNA digested with T~in-lTTT and EcoRI as molecular weight markers.
Figure 6. Diagnostic RE analysis of clones of pBIISKterm. The reacffon products of clones 1 to 5 are shown on the left panel and clones 6 to 10 in the right panel. Three diagnostic reactions were ~elrul,llled for each clone: a) digested with SalI and XbaI (left lane); b) PstI (middle lane); and c) undigested (right lane). The XbaI and SalI double digest releases the 380bp Term fragment as indicated. M is lambda DNA digested with ~inflTTT and EcoRI as molecular weight markers. (Note that undigest DNA for clones 5 and 10 are not shown).
Figure 7. Diagnostic RE analysis of clones of pBl[SK TLVH. The control DNA [pBIISK(BglII) - lanes 1 and 2)] and each clone (1, 3, 6, and 9 - lanes 3 to3Q 8) were digested with SacI (left) and PstI, SalI plus BglII (right lane). Asexpected the SacI digest line~ri.ces the plR.crni-l and the triple digest releases the 950bp LacVH fragment and the 380bp Term fragment as indicated - these fragments are not present in the control DNA. M is la:mbda DNA digested with Hindm and EcoRI as molecular weight markers. Ms is pBluescript plasmid DNA digestedL with HaeIII as molecular weigh.t markers.
CA 0223104~ 1998-03-03 Figure 8. Diagnostic RE analysis of pMOX. Clones 9 and 11 of pMOX were digested with XbaI and ~inrlm (lanes 1 and 2 respectively) which releases the 250bp Pcat fragment that is the same size as an XbaI and ~inrlm digest of pBIISKPcat-Cre (lane 3). Similarly, clones 9 and 11 of pMOX were digested with SalI and PstI (lanes 4 and 5 respectively) which releases the 380bp Term fragment that is the same size as a SalI and PstI digest of pBIISK-TLVH (lane 6). M is lambda DNA digested with ~in~lTTT and EcoRI as molecular weight markers. Ms is pBluescript plasmid DNA digested with Haem as molecular weight markers.
Figure 9. Diagnostic screening for pUX. Twelve clones were digested with PstI (left lane) and undigested (right lane) to screen for the inclusion of the LacVH fragment cloned into pUTcat. Lane C is similarly digested vector pUlcat. Clone 11 clearly has the lacVH fragment cloned into the PstI site since it is releasing a fragment of ~950bp that is a simil~r size to the LacVH
PCR product (lane L) and which the control DNA does not have (left lane C).
It is also clearly evident that the llntligR.sted supercoiled DNA is larger thanthe control DNA (right lane C). M is lambda DNA digested with ~inrlm and EcoRI as molecular weight markers.
Figure 10. Analysis of the orientaffon of the LacVH insert of pUX clone 11.
pUX clones 10, 11 and 12 were digested with BalI (lanes 1, 2 and 3 respectively). A 700bp fragment is released for the correct orientation and 1120bp for the incorrect orientation. Lane 2 clearly intlir:~tRs that the LacVH
fragment in clone 11 is in the correct orientation. pUX clones 10, 11 and 12 were also digested with BglII (lanes 5, 6 and 7 respectively) and in~licRtRs that pUX is linR~ri.sed as expected. M is lambda DNA digested with ~in~m and EcoRI as molecular weight m~rkRrs.
Figure 11. Diagnostic RE analysis of pUX-TT. The isolate clone of pUX-TT
was digested with XhoI (lane 1), SpeI (lane 2), XhoI and SpeI (lane 3) and undigested (lane 5). The double digest releases the 600bp TT heavy chain DNA sequence. Lane 4 is XhoI and SpeI digested pUX. M is lambda DNA
digested with ~in(lm and EcoRI as molecular weight markers.
WO ~7/09436 PCT/AU96/00555 Figure 12. Diagnostic RE analysis of pMOX-TT. T~Telve clones (1 to 12) were digested with SacI and XbaI (left lane) and uncligested (right lane) to screen for the inclusion of the TT light chain DNA firagment cloned into pMOX. Lane C is similarly digested vector pMOX. All clones clearly have 5 the TI fragment cloned into the SacI and XbaI sites since a fragment of ~600bp is released which the control DNA does not have (left lane C). It is also clearly evident that the undigested supercoiled DNA for each clone is larger than the control DNA (right lane C). M is lambda DNA digested with ~inclTTT and EcoRI as molecular weight m~rk~rs.
Figure 13. Diagramatic representations of pMOX (a) and pUX (b).
Figure 14. Diagramatic representation of the propo~ed me-:h~ni.~rn of in vivo recombination between the pMOX and pUX vectors.
Figure 15. Diagramatic representation of the recombinant phage vector pMUX.
EXAMP~.ES
20 Construction of the Lac VH cassette The LacVH cassette was based on the antibocly expression vector MCO1 (Ward et al., 1996), which contains cloning sites for both the heavy and light chain DNA sequences. The first part of the construction of the cassette was to remove the XbaI restriction endonuclease (RE) site (3' cloning 25 site of the light chain) from MCO1. Following digestion with XbaI, MCO1 was treated with klenow fragment of DNA polymerase I (blunted) and subsequently religated and transformed into E. coli strain XLl-blue. Twenty transformants were selected and subjected to RE and gel electrophoretic analysis. Fifteen of these clones were found to be devoid of the XbaI site.
30 One construct, designated MCO4, was retained for further work. The second part of the construction was to remove the light chain leader region from EcoRI to SacI sites. MCO4 was digested with SacI, gel purified and redigested with EcoRI. The double cut MCO4 DNA was then subjected to klenow treatment to 'fill-in' the cohesive ends, ligatecl and transformed i~to 35 XLl-blue. This construction procedure desllvy~d the SacI site but retains theintegrity of the EcoRI site. Twenty transformants were randomly selected CA 0223104~ 1998-03-03 and subjected to RE analysis with EcoRI, SacI and XbaI. At least 10 clones were isolated that displayed the expected profile. This construct is referred toas MC05.
In order to positively determine whether these recombinants were 5 devoid of the 86bp light chain leader sequence, the nucleotide sequence of this region was determined for four clones using MC03.for (MC28) PCR
primer. All four clones were found to be devoid of the said sequence and one clone was retained. Figure 1 indicates that MC05 but not MC01 and MC03 ~which has stuffer fragments cloned into the light and heavy chain cloning 10 sites) are linearized by SacI and XbaI, while all three MCO v~:~Lols are linearized with EcoRI. As expected, it is also evident that MC05 is slightly smaller than MC01.
A 956bp fragment corresponding to position 90 and 987 of MC05 was then amplified by the polymerase chain reaction (PCR) using primers MC19 15 and MC28. The resultant PCR product, designated as the LacVH cassette, contained starting from the 5' end, a unique Pstl site, the lacZ promoter and operator sequence (lacZ P/O), the pel B leader sequence, XhoI and SpeI
unique RE sites, a myc tag, an amber point mutation, a subtilisin open reading frame (ORF), the gene m ORF, a translational stop codon, a mutated 20 loxP site (designated loxP5ll) and a unique BglII site [Figure 2].
The loxp5ll site was introduced into the cassette with the reverse primer MC19, which incorporated a loxP5ll DNA sequence. Intramolecular excision events have been noted where recombination occurs between two loxP sites that are in the same orientation of the DNA substrate (Abremski et 25 al., 1986). In order to avoid this problem in the ffnal construct (pMOX), andin the recombinatorial vector, the loxP511 sequence was positioned in the opposite orientation with repect to the wild type loxP site.
The ~mrlified LacVH cassette was subsequently used in the construction of both the acce~Lor phage vector and the donor plasmid vector.
Construction of pMOX
This vector was based on MC01 and was designed to transiently express 'cre' recombinase until an exr~h~nge between v~cLul~ has occured.
The chloramphenicol acteyltransferase ~CAT) gene promoter (Pcat) is used to 35 drive the cre-recombinase gene t,,~ ssion. Between the Pcat promoter and W O 97/09436 PCT/AU~ 5 the Cre gene is the loxP sequence which is one of tvw designated recombination sites.
Construction of pMOX involved the PCR am~plificatior~ of four discrete fragments that were cloned into MCO1. These fragments are ~ 5 referred to as: Pcat, which contains the chloramphenicol promoter and theloxP site 3' of the promoter; Cre, which contains the sequences necessary to express the creatin recombinase protein; Term, which contains ribonucIease terminator sequences; and LacVH, which contains the cloning site for the heavy chain of an antibody and the loxP511 site 3' of the gene m stop codon.
Construction of the Pcat-Cre cassette The Pcat element was successfully amplifiedL from the CAT gene residing on the plasmid pACYC184 using the primers MC27 and MC17. To determine the efficacy of the amplified Pcat element and whether the loxP
site interfered with transcription, the PCR product was cloned into the promoter-probed vector pklc232-8. Following blunting with Klenow to remove the terminal A's, the Pcat PCR product was dLigested with ~inrlTTT and cloned into the T~in(lmlsmaI site of pkk232-8. The ligation mix was used to transformed XL1-blue cells and promoter efficiency det~rmin~d by spreading the cells on LB agar plates contRining varying (10-50mg) concentrations of chloramphenicol. Five Pcat clones were isolated andL were subjected to PCR
and gel electrophoretic analysis. All five were found to harbour the Pcat element (including the loxP site) indicating that the ]?cat promoter was not compromised because of the presence of the loxP site.
Attempts to clone the Pcat element into the vector pBltSK(BglII) [kindly provided by Dr. Guy Lyons, K~n~m~tsu Laboratory, Universi~ of Sydney] using the unique XbaI and T-TinrlTTT RE sites ~rere unsuccessful. To facilitate cloning of the Pcat element, two further prirn~r.~ were designed (MC50 and MC51) that amplified the Pcat PCR product and also contained additional Hindm RE sites at both ends of the PCR product. The Pcat PCR
fragment and pBltSK~BglII) were digested with T~in~lTrT RE, puri:~ied in an a~garose gel, ligated, and used to transform XL1-blue cells. Twelve transformants were selected and subjected to RE and gel electrophoretic analysis. Eight clones were foumd to contain the Pcat PCR fr~gm~nt and one clone was selected for further work and named pBltS]E~-Pcat. Figure 3 CA 0223104~ 1998-03-03 indicates that the Pcat fragment is released by ~in~lm RE digestion of this plasmid.
The cre recombinase ORF (including the ribosome binding site RBS) was amplified from pBS157 ~kindly provided by Dr Peter Waterhouse, CSIRO
5 Division of Plant Industry, Canberra, Australia] with primers that were designed using the E. coli P1 nucleotide sequence derived by Sternberg et al.
(1986). These two primers (MC25 and MCZ6) incorporated unique ~inrlm and SalI RE sites. After successful ~mplific~tion of the Cre fragment (Figure 4) the product was digested with ~in~lm and SalI, and ligated into ~in~lm 10 and SalI digested pBltSK(BglII). The ligation mix was used to transform XL1-blue cells and eleven of 12 single colonies screened by RE analysis were shown to contain the Cre gene. Four clones further characterised by RE
analysis showed ~he expected b~n-ling patterns (Figure 5). Clone 3 was designated pBltSK-Cre and used for further manipulations.
The Pcat-Cre cassette was constructed by cloning the Pcat fragment (from pBltSK-Pcat) into pBltSK-Cre. This was ~elfolllled by digesting both pBltSK-Pcat and pBltSK-Cre with XbaI and Hin-lm The 4kb fragment from pBltSK-Cre and the 250bp fragment from pBltSK-Pcat were agarose gel purified and ligated The ligation products were used to transform XLl-blue cells and 1Z single colonies were isolated. Initial screening indicated that 10 of the 12 clones had the Pcat fragment cloned into the pBltSK-Cre vector.
Further diagnostic digests were performed on the clones and one was designated pBltSK-PcatCre and used in subsequent manipulation.
Construction of Term-LacVH cassette In order to prevent read through and interference from Pcat, the E.
Coli. 5s ribosomal transcription termin~tor ~rrnbT1T2) [Brosius et al., 19811 was positioned adjacent to the 3' end of the Cre ORF. The rrnbTlT2 t~rmin~tor was successfully amplified from pkk232-8 using the primers MC24 and MC23, generating a product of 340bp. The transcriptional terminator product was restricted with SalI and PstI and ligated into the cloning vector pSP72. The fragment was subsequently extracted from this vector by digestion with SalI and PstI, and ligated into Pstl and SalI digested pBltSK(BglII). The ligation mix was used to transform XL1-blue cells and 12 single colonies were isolated and screened for the inclusion of the Term fragment. All 12 colonies contained the Term fragment (figure 6) and one CA 0223l045 l998-03-03 W O 97/09436 PCT/AU96~ S~' was selected, further characterized and designated pBltSK-Term. This plasmid was used for subsequent manipulations.
The next stage in acceptor phage vector comstruction was to clone the LacVH gene into pBltSK-Term, to produce the Term-LacVH cassette. As - 5 described above a 956bp fragment had been PCR ~mlplified from MC05. New primers (MC48 and MC49) were rlesigned to extend ~he ends of that fragment, adding ~intlm cloning sites to both ends. A fragment of a~ ;ate size was ~mplifi~hle using these two primers and the original PCR product as template. The PCR product and pBllSK-Term were then digested with PstI and BglII, gel purified and ligated. The ligation reaction was used to transform XL1-blue cells and 12 single colonies were isolated and screened for the inclusion of the LacVH fragment. Eleven of the 12 colonies contained the LacVH gene. Further RE rliRgrnostic digests were performed (Figure 7) and one clone was selected andl designated pBltSK-TLVH. This clone was used for subsequent DNA manipulations.
Construction of pMOX
The final step in the construction of pMOX involved digesting: a) MC01 with XbaI and BglII; b) pBltSK-TLVH with B~slII and SalI; and c) pBltSK-PcatCre with SalI and XbaI. The MC01 vect~r, TLVH and PcatCre cassettes were purified in an agarose gel, mixed and ligated. The ligation mix was used to transform XLl-Blue cells and 12 colonies were isolated and screened for the correct construction. Figure 8 inflic~t~s expected restrictio digest patterns of the resultant pMOX vector.
Construction of pUX
Construction of pUX was based on pUC19 (Yanisch et al., 1985).
Since both the donor and acce~lor vectors possessed the same antibiotic resistance (~mpir,illin - ApR) determin~nt, the ApR gene of pUC19 was replaced with a PCR amplified Tetracyclin resistance (TcR). The entire TcR
gene was amplified from pACYC184, using primers that included PvuI RE
sites at both ends of the PCR product (MC21 and MC22). The TcR gene was restricted with PvuI, and ligated with the 179obp Pvu~ restricted fragment of pUC19. This fragment was partially devoid of the ApR coding region (thereby m~king it sensitive to Ap), as well as the cod~ing region for lacZ. Theligation ~ cLul~ was then transformed into the Tc E. coli sensitive strain CA 0223l04~ l998-03-03 W O 97/09436 PCT/AU9~ 'C' NM522, and recombinants were selected for resistance to Tc. Following PvuI
RE analysis one clone (designated pUT-1, 3209bp) was retained.
The reIn~inrle.r of the vector was constructed by cloning two PCR
generated fragments into the MCS of pUC19. These fragments are referred to as: CAT (which contains the sequences that induce resistance to chloramphenicol); and LacVH (which contains the cloning site for the heavy chain of an antibody).
The chlor~mph~ir.ol resistance gene (the ORF excluding its promoter region) was PCR amplified using primers MC18 and MC20. The cat gene primers were based on the nucleotide sequence of the cat gene of the plasmid Tn9 (Alton and Vapnek, 1979). After amplifcation the loxP-cat gene was restriced with SacI/PstI and ligated into SacIlPstI restricted pl~T-1 vector.
The ligation ~ e was transformed into NM5Z2 and mini-plasmid lysate preps of eighteen randomly chosen isolates were analysed by RE and gel electrophoresis. Additional characterization of four clones with PCR
revealed that all four clones (designated pl~Tcat) contained, the loxP-cat gene.
The next step in the construction of pUX was to clone the lacVH gene into the Psff site. This was accomplished by amplifying the LacVH PCR
fragment described earlier with primers (MC48 and MC58) that added a PstI
site to both ends of the LacVH gene. The PCR fragment and pUTcat was restricted with PstI, the DNA was purified in an agarose gel and the DNA
ligated. The ligation mixture was transformed into NM522 cells and mini-plasmid lysate preps of eighteen randomly isolated clones were analysed by RE and gel electrophoresis. One clone contained the DNA insert (Figure 9) which, with additional RE analysis, was shown to be in the correct orientation (Figure 10). This clone was rlesign~ted pUX.
~ vivo recombination of pUX and pMOX
To test for 'in vivo' recombination between pUX and pMOX, the heavy chain of a tetanus toxoid (~) Fab was cloned into pUX, while the light chain was cloned into pMOX. Phage was then prepared from pMOX-TT, and was used to infect log phase HB2151 cells previously transfected with pUX-TI.
Construction of pUX-TT and pMOX-TT
CA 0223l045 l998-03-03 W O 97/09436 PCT/AU~
The tetanus toxoid heavy chain was RE digested with XhoVspeI and purified in an agarose gel from MC01-TT. This frag;ment was ligated into - XhoVspeI RE digested pUX. The ligation llPLcLIlre was used to transform HB2151 cells and a single colony was isolated. This; colony was screened using RE digests and gel electrophoresis for the inclusion of the TT heavy chain DNA. Figure 11 indicates that this clone contained the IT heavy chain DNA sequences. The tetanus toxoid light chain was, RE digested with SacI/XbaI and purified in an agarose gel from MC01-TT. This fragment was ligated into SacVXbaI RE digested pMOX. The ligation ~ re was used to 0 transform XL1-blue cells and 1Z colonies were isolated and screened using RE digests and gel electrophoresis for the inclusion of the TT light chain.
Figure 12 indicates that all 12 colonies contained the TT light chain DNA
sequences. These two plasmids were referred to as pUX-lT and pMOX-TT.
Preparation of pMOX-TT phage.
pMOX-TT was transformed into XL1-blue and 250,u1 of an overnight culture was used to inoculate 2YT cont~ining 2% glucose, carb~nicillin (carb -50~Lg/ml)tetracycline (tet - 1o,ug/ml) and then rescue with helper phage (VCS-M13). The culture was incubated for 2 hours at 37~C with shaking and ZO thencentrifuged at room L~nlyeltlture at 4500rpm for 15 minutes. The bacterial pellet was resuspended in 50ml of 2YT containing carb 50,ug/ml, tetlO~lg/ml and k~n~mycin 70~1g/ml and incubated overnight at 37~C with sh~king. The bacteria were pelleted by centrifugation at 8000rpm at room temperature, The phage were precipitated from the supernatant by ~flrling 1/5vol 20~ PEG6000/2.5M NaCl, mixing and leaving on ice with occasional sh~king for 1 hour. The phage were collected by cenltrifugation at 8000rpm for 20 lminutes at 4~C. The pellet was resuspended in 500,ul of 19~o BSA, and bacterial debris was removed by a high speed centrifugation in a microfuge tube and stored at 4~C.
k2 vivo recombination between pUX-TT and pMOX-lT
A 1ml culture of pUX transformed HB2151 cells in media cont~ining tetracycline (OD600 = 0.8) were infected with lx109 pMOX-~ phage a~d allowedL to stand at 37~C for 30 minutes. Carb (2511g/rnl) was added and the culture incubated for 30 minutes with sh~king. Further carb (25,u~/ml) was added and incubated with .ch~king for a further 3 hours. An ali~uot of cells CA 0223l045 l998-03-03 added and incubated with shaking for a further 3 hours. An aliquot of cells (250,u1) was obtained 1 and 4 hours after infection and plated onto: a) Carb;
b) Tet; c) ChlorlO; d) Chlor50; e) Carb/ChlorlO; and f) Carb/Chlor50.
Since pMOX is tet sensitive and pUX is carb sensitive, growth of cells 5 in culture indicated that the cells contain both pMOX and pUX.
Chloramphenicol further selects for cells that have recombinants contRining the pMUX vector since chlor~mrh~ir.ol resistance is acquired only after recombination. A lawn of colonies were observed on both chlor and carb/chlor plates (at both concentrations of chlor) following infection of 10 pMOX-TT into the culture contRining HB2151/pUX-TT indicating that recombination had occurred.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the 15 invention as broadly described. The present embodiments are, th~le:rule, to be considered in all respects as illustrative and not restrictive.
List of Primers MC03.for (MC28) 5'.. ACC TGC AGA ACA GTG AGC GCA ACG CAA T.. 3' Pstl site for cloning MC03.rev (MC19) 5'..GAA GAT CTT C~AT AAC TTC GTA TAT ACA TAT
GTA TAC GAA GTT ATlG CGG CCG CTT AAA TTA AIT AGC..3' BglII site for cloning and ~loxP511] recombination site 25 Pcat.for (MC50) 5'CCC AAG Cl~ GGG CTC TAG AGC CGA CGC ACT TTG
CGC CGA AT..3' Forward primer for Pcat PCR fragment; XbaI and ~dm PcatPCR.rev (MC5 1) 5 '. .CCC AAG CTI CCC AAG C~T GGG ATA ACT TCG
TAT AGC ATA CAT TAT3' Reverse primer for Pcat PCR fragment with LoxP site; HindIII.
Pcat.for (MC27) 5'..GCT CTA GAG CCG ACG CAG TTT GCG CCG A11..3' Reverse primer for the ~mrlific~tion of LacVH of pMOX.
Pcat.rev (MC17) 5'..GGG AAG C~ CCC ~ATA ACT TCG TAT AGC ATA
CAT TAT ACG AAG TTA TlTC GAT AAC TCA AAA AAT ACG
CC.. 3' Reverse PCR primer for ~mplifir:~tion of the CAT gene lol~r fro Tn9. HindIII for cloning and ~loxPl Cre.rev (MC25) 5'..ACG CGT CGA CCG CGT TAA TGG CTA ATC GC..3' Reverse primer for the amplification of cre recombinase of pMOX.
Cre.for (MC26) 5'..CCC AAG CTT CTG AGT GTT AAA TGT CCA ATT
TAC.. 3' Forward primer for the ~mplific~tic ~ of cre recombinase of pMOX.
CA 0223l045 l998-03-03 WO97/09436 PCT/AU9~ 555 Term.for (MC24) 5'..ACG CGT CGA CCA GAA GTG AAA CGC CGT A..5' Forward primer for the ~mplification of terlrnin~tnr for pMOX.
Term.rev (MC23) 5'..TTC TGC AGT TCC TGA TGA TGC AAA AAC GAG
GC..3' Reverse primer for the ~mplificatiorl of t~rmin~tor of pMOX.
5 MCO5.for (MC48) 5'..CCC AAG c7'r GGG ACC TGC AGA ACA GTG AGC
GCA ACG CAA T. . 3' Forward primer for P('R of LacVH of pMOX;
PslI, ~ndIII
MCO5.rev (MC49) 5'..CCC AAG C7T GGG AAG ATC TTC ATA ACT TCG
TAT ATA CAT A..3' Reverse primer for PClR of LacVH with loxP
site; BglII, HindIII.
tet.rev ~MC21) 5'..TAC GAT CGT ATT CAC AGT TCT CCG CAA GA..3' Reverse primer for the ~mplifir:~tion of tetR for pUX.
tet.for (MC22) 5'..ATC GAT CGA TCA AAT GTA GCA CCT GAA GTC AG..3' Forward primer for the ~mplification of tetR for pUX.
5 cat.for (MC18) 5'..CGA GCI'CGrA TAA CTT CGT A'~A ATG TAT GCT ATA
CGA AGT TATl GAT TTT CCA GGA GCT~AG GAA..3' Forward PCR primer for the ~mplific~tion of the CAlr resistance gene from Tn9; SacI, [lo~Pl cat.rev (MC20) 5'..TIT GCA GAT CGT CAA TTA CCT CCA CG..3' Reverse Z0 primer for RmplifiC~tion of CatR gene for pUX.
MCO.rev PPCR (MC58~ 5'..AAA ACT GCA GCC AAG AllC ITC ATA ACT
TCG TAT ATA CAT A..3' Reverse PCR prinner for LacVH MCO PC~
product; Pstl, BglII
J CA 0223104~ 1998-03-03 ._. ~ - ' ~ -. ~
_ . . ~ ,, BIBLIOGRAPHY
Abremski, K., Frommer, B. and Hoess, R. H. (19~6) T.inking-Ilumber changes in the DNA substrate during Cre-mediated loxP site-specific recombination.
Journal of Molecular Biology 192(1):17-26.
Alton, N. K. and Vapnek, D. (1979) Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9. Naf.ure 28Z(5741):864-9.
Brosius, J., Dull, T. J., Sleeter, D. D. and Noller, E3:. F. (1981) Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. Journal of Molecular Biology 148(2):107-27.
Geoffroy, R., Sodoyer. R, and Aujame, L. (1994) A new phage display system to construct multicombinatorial libraries of very large antibody repertoires.
Gene. 151:109-113.
Griffiths, A. D., Williams, S.C., Hartley, O, Tomlinson, I.M., Waterhouse, P., Crosby, W.L, Kontermann, R,E., Jones, P.T., Low, N.M., Allison, T.J (1994) Isolation of high affinity hurnan antibodies directly from large synthetic repertoires. Embo Journal. 13 :3245-60.
Sternberg, N., Sauer, B., Hoess, R. and Abremski, K. (1986) Bacteriophage P1 cre gene and its regulatory region. Evidence for multiple promoters and for regulation by DNA methylation. Journal of Molecular Biology 187(2): 197-Z 12.
Ward, R. L., Clark, M. A., Lees, J., Hawkins, N. J. (1996) Retrieval of human antibodies from phage-display libraries using enzymatic clevage. J.
Immunol. Methods. 189: 73-82.
Waterhouse, P., Griffiths A.D., Johnson, K.S., and. Winter. G. (1993) Combinatorial infection and in vivo recombination: a strategy for m;~king large phage antibody repertoires. Nucleic Acids Research. 21:2265-6.
Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUClg vectors. Gene 33(1):103-19.
;HEET
~P~/AlJ
Claims (26)
1. A method for producing a phage display vector, which method includes causing or allowing recombination between (i) a first vector which includes a sequence encoding a first polypeptide chain of a specific binding pair member; and (ii) a second phage vector which includes a sequence encoding Cre recombinase operatively linked to a control sequence which allows expression of the cre recombinase and a sequence encoding a second polypeptide chain of a specific binding pair member;
wherein either the first or second polypeptide chain is fused to a component of a replicable genetic display package which thereby displays the fused polypeptide at the surface of replicable genetic display packages;
and wherein the recombination event gives rise to a recombinant phage vector which includes sequences encoding both the first and second polypeptide chains and in which expression of the Cre recombinase is substantially inhibited.
wherein either the first or second polypeptide chain is fused to a component of a replicable genetic display package which thereby displays the fused polypeptide at the surface of replicable genetic display packages;
and wherein the recombination event gives rise to a recombinant phage vector which includes sequences encoding both the first and second polypeptide chains and in which expression of the Cre recombinase is substantially inhibited.
2. A method according to claim 1 wherein the second phage vector includes a nucleic acid sequence encoding a promoter sequence, a first recombination site downstream and adjacent to the promoter sequence and an open reading frame for cre-recombinase positioned downstream and adjacent to the recombination site, such that the promoter allows expression of the Cre recombinase sequence; and wherein recombination takes place at the first recombination site such that the open reading frame for Cre recombinase is separated from the promoter sequence.
3. A method according to claim 2 wherein the promoter sequence is derived from a promoter for a gene encoding a selectable marker.
4. A method according to claim 3 wherein the selectable marker is a gene encoding resistance to an antimicrobial agent.
5. A method according to claim 4 wherein the antimicrobial agent is chloramphenicol.
6. A method according to any one of claims 1 to 5 wherein the first vector includes a first recombination site upstream and adjacent to a sequence encoding an open reading frame of a selectable marker, wherein recombination results in the positioning of the sequence encoding an open reading frame of a selectable marker adjacent to and downstream of the promoter sequence such that the promoter sequence allows expression of the selectable marker.
7. A method according to claim 6 wherein the first vector includes a terminator sequence upstream and adjacent to the first recombination site.
8. A method according to any one of claims 1 to 7 wherein the first and second vectors each include a first recombination site and a second recombination site different from the first, site-specific recombination taking place between first recombination sites on the first and second vectors and between second recombination sites on the first and second vectors, but not between the first recombination site and the second recombination site on the same vector.
9. A method according to any one of claims 2 to 8 wherein the first recombination site is the loxP sequence.
10. A method according to claim 8 or claim 9 wherein the second recombination site is a mutant loxP sequence.
11. A method according to claim 10 wherein the second recombination site is the loxP511 sequence.
12. A method according to any one of claims 1 to 11 wherein the first vector is a plasmid.
13. A method according to any one of claims 1 to 12 wherein recombination takes place in a bacterial host and the recombinant phage particles are isolated from the bacterial host.
14. A method according to any one of claims 1 to 13 wherein the specific binding pair member is an antibody or an antibody fragment.
15. A method according to any one of claims 1 to 14 wherein either the first or second vector further includes (a) a sequence encoding a detectable label;
(b) an inducible stop codon; and (c) a sequence encoding an enzymatic cleavage site such that the sequences described in (a) to (c) are included in the recombinant phage vector.
(b) an inducible stop codon; and (c) a sequence encoding an enzymatic cleavage site such that the sequences described in (a) to (c) are included in the recombinant phage vector.
16. A phage vector which includes a nucleic acid sequence encoding a promoter sequence, a first recombination site downstream and adjacent to the promoter sequence and an open reading frame for Cre recombinase positioned downstream and adjacent to the recombination site such that the promoter drives expression of the cre-recombinase.
17. A phage vector according to claim 16 which further includes at least one nucleic acid sequence encoding a polypeptide chain of a specific binding pair member.
18. A phage vector according to claim 17 wherein the specific binding pair member is an antibody or an antibody fragment.
19. A phage vector according to any one of claims 16 to 18 wherein the promoter sequence is derived from a promoter for a gene encoding a selectable marker.
20. A phage vector according to claim 19 wherein the selectable marker is a gene encoding resistance to an antimicrobial agent.
21. A phage vector according to claim 20 wherein the antimicrobial agent is chloramphenicol.
22. A phage vector according to any one of claims 16 to 21 wherein the recombination site is the loxP sequence.
23. A phage vector according to any one of claims 16 to 22 wherein the vector further includes a second recombination site which is different to the first recombination site.
24. A phage vector according to claim 23 wherein the second recombination site is a mutant of the loxP sequence.
25. A phage vector according to claim 24 wherein the second recombination site is the loxP511 sequence.
26. A phage vector according to any one of claims 16 to 25 wherein the vector further includes (a) a sequence encoding a detectable label;
(b) an inducible stop codon; and (c) a sequence encoding an enzymatic cleavage site.
(b) an inducible stop codon; and (c) a sequence encoding an enzymatic cleavage site.
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| US6720140B1 (en) | 1995-06-07 | 2004-04-13 | Invitrogen Corporation | Recombinational cloning using engineered recombination sites |
| EP0937098B1 (en) | 1995-06-07 | 2002-08-14 | Invitrogen Corporation | Recombinational cloning in vitro using engineered recombination sites |
| US6143557A (en) * | 1995-06-07 | 2000-11-07 | Life Technologies, Inc. | Recombination cloning using engineered recombination sites |
| US6051409A (en) | 1995-09-25 | 2000-04-18 | Novartis Finance Corporation | Method for achieving integration of exogenous DNA delivered by non-biological means to plant cells |
| US5851808A (en) | 1997-02-28 | 1998-12-22 | Baylor College Of Medicine | Rapid subcloning using site-specific recombination |
| WO1999021977A1 (en) | 1997-10-24 | 1999-05-06 | Life Technologies, Inc. | Recombinational cloning using nucleic acids having recombination sites |
| WO1999025855A1 (en) | 1997-11-18 | 1999-05-27 | Pioneer Hi-Bred International, Inc. | Mobilization of viral genomes from t-dna using site-specific recombination systems |
| US7102055B1 (en) | 1997-11-18 | 2006-09-05 | Pioneer Hi-Bred International, Inc. | Compositions and methods for the targeted insertion of a nucleotide sequence of interest into the genome of a plant |
| DE69831265T2 (en) | 1997-11-18 | 2006-06-08 | Pioneer Hi-Bred International, Inc. | COMPOSITIONS AND METHODS FOR THE GENETIC MODIFICATION OF PLANTS |
| WO1999025840A1 (en) | 1997-11-18 | 1999-05-27 | Pioneer Hi-Bred International, Inc. | A novel method for the integration of foreign dna into eukaryoticgenomes |
| JP2002522029A (en) | 1998-07-27 | 2002-07-23 | ジェネンテック・インコーポレーテッド | Improved transformation efficiency in phage display by modification of coat protein |
| NZ525134A (en) | 1999-03-02 | 2004-09-24 | Invitrogen Corp | Compositions and methods for use in recombinational cloning of nucleic acids |
| US7198924B2 (en) | 2000-12-11 | 2007-04-03 | Invitrogen Corporation | Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites |
| US7560622B2 (en) | 2000-10-06 | 2009-07-14 | Pioneer Hi-Bred International, Inc. | Methods and compositions relating to the generation of partially transgenic organisms |
| US20030235814A1 (en) * | 2002-06-19 | 2003-12-25 | Los Alamos National Laboratory | Compositions and methods for selecting open reading frames |
| JP2007512838A (en) | 2003-12-01 | 2007-05-24 | インヴィトロジェン コーポレーション | Nucleic acid molecules containing recombination sites and methods of use thereof |
| DK2189539T4 (en) | 2008-11-21 | 2018-09-17 | Chimera Biotec Gmbh | Conjugate complexes for analyte detection |
| US9712606B2 (en) * | 2014-05-21 | 2017-07-18 | Nasdaq Technology Ab | Efficient and reliable host distribution of totally ordered global state |
| CN110100000B (en) | 2016-12-26 | 2023-06-20 | Jcr制药股份有限公司 | BDNF-containing fusion proteins |
| KR102497564B1 (en) | 2016-12-26 | 2023-02-07 | 제이씨알 파마 가부시키가이샤 | Novel anti-human transferrin receptor antibody capable of penetrating blood-brain barrier |
| KR20200116952A (en) | 2018-02-05 | 2020-10-13 | 제이씨알 파마 가부시키가이샤 | Method for delivering drugs to the muscles |
-
1995
- 1995-09-05 AU AUPN5239A patent/AUPN523995A0/en not_active Abandoned
-
1996
- 1996-09-05 WO PCT/AU1996/000555 patent/WO1997009436A1/en not_active Application Discontinuation
- 1996-09-05 EP EP96928283A patent/EP0850309A1/en not_active Withdrawn
- 1996-09-05 JP JP9510704A patent/JPH10511001A/en active Pending
- 1996-09-05 CA CA002231045A patent/CA2231045A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| JPH10511001A (en) | 1998-10-27 |
| EP0850309A1 (en) | 1998-07-01 |
| AUPN523995A0 (en) | 1995-09-28 |
| WO1997009436A1 (en) | 1997-03-13 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |