WO1997009436A1 - Procede de production de phages vecteurs de presentation - Google Patents

Procede de production de phages vecteurs de presentation Download PDF

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Publication number
WO1997009436A1
WO1997009436A1 PCT/AU1996/000555 AU9600555W WO9709436A1 WO 1997009436 A1 WO1997009436 A1 WO 1997009436A1 AU 9600555 W AU9600555 W AU 9600555W WO 9709436 A1 WO9709436 A1 WO 9709436A1
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WIPO (PCT)
Prior art keywords
sequence
recombination
vector
recombination site
promoter
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PCT/AU1996/000555
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English (en)
Inventor
Tony Vancov
David Zahra
Nicholas John Hawkins
Robyn Lynne Ward
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Crc For Biopharmaceutical Research Pty. Ltd.
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Priority to JP9510704A priority Critical patent/JPH10511001A/ja
Priority to AU67823/96A priority patent/AU6782396A/en
Priority to EP96928283A priority patent/EP0850309A1/fr
Publication of WO1997009436A1 publication Critical patent/WO1997009436A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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)

Definitions

  • the present invention relates to phage display vectors and to methods for producing recombinant phage display vectors.
  • Functional antigen-binding domains of antibodies can be displayed on the surface of filamentous bacteriophage.
  • International patent application WO 93/19172 describes methods, recombinant host cells and kits for production of antibodies displayed on the surface of phage.
  • recombination occurs between two vectors comprising nucleic acid encoding immunoglobulin 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 panning.
  • the size of antibody libraries generated using most phage display systems is limited by the low transformation efficiency of Escherichia coli.
  • 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 exceptionally larger numbers. Two different mechanisms of site-specific recombination have been used to achieve the association of the two libraries: l.
  • the lox-Cre system of bacteriophage Pl Waterhouse et al., 1993;
  • E.coli is transformed with a repertoire of heavy chain antibody genes (encoded on a phagemid).
  • Cre recombinase is provided in vivo by infecting the E.coli with Pl, 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) 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 repertoire of light chain antibody 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.
  • a major problem of the methods used to date is that the recombination process is reversible, therefore stable recombinants are not 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.
  • the present invention consists in a method for producing a phage display vector, which method includes causing or allowing recombination between
  • 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.
  • 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.
  • the promoter sequence is derived from a promoter for a gene encoding a selectable marker.
  • the selectable marker is a gene encoding resistance to an anti
  • the antimicrobial agent is chloramphenicol.
  • the promoter is an inducible promoter.
  • the inducible promoter may provide increased control over initiation of the recombination event.
  • the second phage vector also includes a terminator sequence positioned downstream of the Cre recombinase open reading frame in order to prevent undesired transcription of genes downstream of the Cre recombinase gene.
  • the terminator may be the rrnbTlT2 terminator sequence.
  • 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 the open reading frame of a selectable marker on the recombinant phage vector adjacent to and downstream of the promoter sequence such that the promoter sequence allows expression of the selectable marker.
  • 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 preferred embodiment the selectable marker is a gene encoding resistance to an antimicrobial agent.
  • the antimicrobial agent is chloramphenicol.
  • 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.
  • the first recombination site is the loxP sequence.
  • the second recombination site is preferably a mutant loxP sequence, more preferably the i ⁇ xP ⁇ ll sequence.
  • 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.
  • a terminator sequence may be included upstream of the recombination site of the first vector used in the method of the present invention. This terminator sequence should prevent any undesirable expression of the Cre recombinase following the recombination event.
  • This terminator sequence is preferably different to the terminator sequence, if present, on the original second phage vector.
  • the terminator sequence may, for example, be the ⁇ terminator.
  • the recombinant phage particles may be isolated from the host organism.
  • either the first or second vector further includes
  • the detectable label is the c-myc peptide label
  • the inducible stop codon is the amber codon
  • the enzymatic cleavage site is the subtilisin cleavage site. It will be appreciated that the detectable label allows immunological detection of expressed antibodies without the need to use antibodies against 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 nonsupressor strains oi E.coli for expression.
  • the enzymatic cleavage site facilitates release of the phage from the specific binding pair member.
  • the first vector is pUX-1 and the second phage vector is pMOX-1.
  • 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.
  • the promoter sequence is derived from a promoter for a gene encoding a selectable marker.
  • the selectable marker is a gene encoding resistance to an antimicrobial agent. More preferably, the antimicrobial agent is chloramphenicol.
  • the recombination site is the loxP sequence.
  • the vector further includes a second recombination site which is different to the first recombination site.
  • the second recombination site is preferably a mutant of the loxP sequence, more preferably the 7 ⁇ xP511 sequence.
  • the method of the present invention may be used to produce phage display vectors which are suitable for preparing combinatorial libraries of antibodies.
  • the specific binding pair member may therefore be an antibody or an antibody fragment.
  • the first polypeptide chain may, for example, be an immunoglobulin heavy chain and the second polypeptide chain may be an immunoglobulin light chain, or vice versa.
  • the phage vector used in the method of the present invention is designed to transiently express the Cre recombinase until exchange between the two vectors occurs.
  • the promoter is used to drive Cre recombinase gene expression until recombination occurs.
  • the recombination event substantially silences expression of the Cre recombinase and therefore gives rise to a stable recombinant phage vector.
  • the recombination event results in an exchange of 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.
  • 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 containing recombinant phage vectors.
  • FIG. 1 Diagnositic RE analysis of MC05. Lanes 1 to 4 are undigested MC05 clones 1 and 2, MCOl and MC03 respectively. Lanes 5 to 8 are Sad digested MC05 clones 1 and 2, MCOl and MC03 respectively and indicates that MCO ⁇ remains undigested whereas MCOl and MC03 are linearised. Lanes 10 to 13 are Xbal digested MC05 clones 1 and 2, MCOl and MC03 respectively and indicates that MC05 remains undigested whereas MCOl and MC03 are linearised. Lanes 14 to 17 are EcoRI digested MC05 clones 1 and 2, MCOl and MCO3 respectively and indicates that all plasmids are linearised with this RE. Lane 9 is lambda DNA digested with Hindlll and EcoRI as molecular weight markers.
  • Lane 2 PCR amplification of the LacVH cassette from MCO5.
  • 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 MCO3 produces a product of 2.5kb as expected.
  • Lane 1 is lambda DNA digested with HindDI and EcoRI as molecular weight markers.
  • Lanes 1 to 9 are mini prep clones 3, 4, 6 to 12 of pBLISK-Pcat digested with Bglll and Sail.
  • C is pB ⁇ SK(BglII) digested with Bgi ⁇ and Sail. The banding pattern indicates that all clones except clone 11 has the 250bp Peat fragment cloned into pBHSK(Bgi ⁇ ).
  • M is lambda DNA digested with HindHI and EcoRI as molecular weight markers.
  • Ms is pBluescript plasmid DNA digested with Haem as molecular weight markers.
  • FIG. 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 ⁇ lkb as expected.
  • M is lambda DNA digested with Hindi ⁇ and EcoRI as molecular weight markers.
  • Figure 5 Diagnostic RE analysis of clones of pBHSKcre.
  • C is the control DNA [pB ⁇ S (Bgi ⁇ )] digested with BssHII and Xbal.
  • Lanes 3, 4, 5, and 7 are the respective clones of pBIISKcre digested with BssHII and Xbal as indicated.
  • An Xbal digest was expected to linearise the control and clones.
  • the expected fragment sizes for the pBssHH RE digest were 2.8kb, 861bp and
  • Figure 6 Diagnostic RE analysis of clones of pBHSKterm.
  • the reaction 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 performed for each clone: a) digested with Sail and Xbal (left lane); b) PstI (middle lane); and c) undigested (right lane).
  • the Xbal and Sail double digest releases the 380bp Term fragment as indicated.
  • M is lambda DNA digested with Hindlll and EcoRI as molecular weight markers. (Note that undigest DNA for clones 5 and 10 are not shown).
  • FIG. 7 Diagnostic RE analysis of clones of pBHSK-TLVH.
  • the control DNA [pBIISK(Bgi ⁇ ) - lanes 1 and 2)] and each clone (1, 3, 6, and 9 - lanes 3 to 8) were digested with Sad (left) and PstI, Sail plus Bglll (right lane).
  • the Sad digest linearises the plasmid 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 lambda DNA digested with HindHI and EcoRI as molecular weight markers.
  • Ms is pBluescript plasmid DNA digested with HaeDI as molecular weight markers.
  • Clones 9 and 11 of pMOX were digested with Xbal and HindHI (lanes 1 and 2 respectively) which releases the 250bp Peat fragment that is the same size as an Xbal and HindHI digest of pBHSKPcat-Cre (lane 3).
  • clones 9 and 11 of pMOX were digested with Sail and PstI (lanes 4 and 5 respectively) which releases the 380bp Term fragment that is the same size as a Sail and PstI digest of pBHSK-TLVH (lane 6).
  • M is lambda DNA digested with HindHI and EcoRI as molecular weight markers.
  • Ms is pBluescript plasmid DNA digested with HaeBJ 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 pUTcat. Clone 11 clearly has the lacVH fragment cloned into the PstI site since it is releasing a fragment of ⁇ 950bp that is a similar size to the LacVH
  • M is lambda DNA digested with HindHI and EcoRI as molecular weight markers.
  • FIG. 10 Analysis of the orientation of the LacVH insert of pUX clone 11.
  • pUX clones 10, 11 and 12 were digested with Ball (lanes 1, 2 and 3 respectively). A 700bp fragment is released for the correct orientation and 1120bp for the incorrect orientation. Lane 2 clearly indicates that the LacVH fragment in clone 11 is in the correct orientation.
  • pUX clones 10, 11 and 12 were also digested with BglH (lanes 5, 6 and 7 respectively) and indicates that pUX is linearised as expected.
  • M is lambda DNA digested with HindHI and EcoRI as molecular weight markers.
  • Figure 11 Diagnostic RE analysis of pUX-TT.
  • the isolate clone of pUX-TT was digested with Xhol (lane 1), Spel (lane 2), Xhol and Spel (lane 3) and undigested (lane 5). The double digest releases the 600bp TT heavy chain DNA sequence.
  • Lane 4 is Xhol and Spel digested pUX.
  • M is lambda DNA digested with HindHI and EcoRI as molecular weight markers.
  • Figure 12 Diagnostic RE analysis of pMOX-TT. Twelve clones (1 to 12) were digested with Sad and Xbal (left lane) and undigested (right lane) to screen for the inclusion of the TT light chain DNA fragment cloned into pMOX.
  • Lane C is similarly digested vector pMOX. All clones clearly have the TT fragment cloned into the Sad and Xbal 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 HindlH and EcoRI as molecular weight markers.
  • Figure 13 Diagramatic representations of pMOX (a) and pUX (b).
  • Figure 14 Diagramatic representation of the proposed mechanism of in vivo recombination between the pMOX and pUX vectors.
  • Figure 15 Diagramatic representation of the recombinant phage vector pMUX.
  • the LacVH cassette was based on the antibody expression vector MCOl (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 Xbal restriction endonuclease (RE) site (3' cloning site of the light chain) from MCOl. Following digestion with Xbal, MCOl 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 Xbal site.
  • RE restriction endonuclease
  • MCO4 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 Sad sites. MC04 was digested with Sad, gel purified and redigested with EcoRI. The double cut MC04 DNA was then subjected to klenow treatment to 'fill-in' the cohesive ends, ligated and transformed into XLl-blue. This construction procedure destroyed the Sad site but retains the integrity of the EcoRI site. Twenty transformants were randomly selected and subjected to RE analysis with EcoRI, Sad and Xbal. At least 10 clones were isolated that displayed the expected profile. This construct is referred to as MC05.
  • a 956bp fragment corresponding to position 90 and 987 of MC05 was then amplified by the polymerase chain reaction (PCR) using primers MC19 and MC28.
  • the resultant PCR product designated as the LacVH cassette, contained starting from the 5' end, a unique PstI site, the lacZ promoter and operator sequence (lacZ P/O), the pel B leader sequence, Xhol and Spel unique RE sites, a myc tag, an amber point mutation, a subtilisin open reading frame (ORF), the gene HI ORF, a translational stop codon, a mutated loxP site (designated loxP ⁇ ll) and a unique BglH site [ Figure 2].
  • the loxP ⁇ ll site was introduced into the cassette with the reverse primer MC19, which incorporated a loxP ⁇ ll 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 al., 1986). In order to avoid this problem in the final construct (pMOX), and in the recombinatorial vector, the loxP ⁇ ll sequence was positioned in the opposite orientation with repect to the wild type loxP site.
  • the amplified LacVH cassette was subsequently used in the construction of both the acceptor phage vector and the donor plasmid vector.
  • This vector was based on MCOl and was designed to transiently express 'cre' recombinase until an exchange between vectors has occured.
  • the chloramphenicol acteyltransferase (CAT) gene promoter (Peat) is used to drive the cre-recombinase gene expression. Between the Peat promoter and the Cre gene is the loxP sequence which is one of two designated recombination sites.
  • the Peat element was successfully amplified from the CAT gene residing on the plasmid pACYCl84 using the primers MC27 and MC17. To determine the efficacy of the amplified Peat element and whether the loxP site interfered with transcription, the PCR product was cloned into the promoter-probed vector pkk232-8. Following blunting with Klenow to remove the terminal A's, the Peat PCR product was digested with HindHI and cloned into the HindHI/Smal site of pkk232-8. The ligation mix was used to transformed XLl-blue cells and promoter efficiency determined by spreading the cells on LB agar plates containing varying (10-50mg) concentrations of chloramphenicol.
  • Peat clones Five Peat clones were isolated and were subjected to PCR and gel electrophoretic analysis. All five were found to harbour the Peat element (including the loxP site) indicating that the Peat promoter was not compromised because of the presence of the loxP site. Attempts to clone the Peat element into the vector pBltSK(BglH)
  • the cre recombinase ORF (including the ribosome binding site RBS) was amplified from pBS157 [kindly provided by Dr Peter Waterhouse, CSIRO Division of Plant Industry, Canberra, Australia] with primers that were designed using the E. coli Pl nucleotide sequence derived by Sternberg et al. (1986). These two primers (MC25 and MC26) incorporated unique HindlH and Sail RE sites. After successful amplification of the Cre fragment ( Figure 4) the product was digested with HindlH and Sail, and ligated into HindlH and Sail digested pBltSK(BglH).
  • the E. Coli. 5s ribosomal transcription terminator (rrnbTlT2) [Brosius et al., 1981] was positioned adjacent to the 3' end of the Cre ORF.
  • the rrnbTlT2 terminator was successfully amplified from pkk232-8 using the primers MC24 and MC23, generating a product of 340bp.
  • the transcriptional terminator product was restricted with Sail and PstI and ligated into the cloning vector pSP72.
  • the fragment was subsequently extracted from this vector by digestion with Sail and PstI, and ligated into PstI and Sail digested pBltSK(BglH).
  • the ligation mix was used to transform XLl-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 was selected, further characterized and designated pBltSK-Term. This plasmid was used for subsequent manipulations.
  • the next stage in acceptor phage vector construction was to clone the LacVH gene into pBltSK-Term, to produce the Term-Lac VH cassette.
  • a 956bp fragment had been PCR amplified from MCO ⁇ .
  • New primers (MC48 and MC49) were designed to extend the ends of that fragment, adding HindHI cloning sites to both ends.
  • a fragment of appropriate size was amplifiable using these two primers and the original PCR product as template.
  • the PCR product and pBltSK-Term were then digested with PstI and BglH, gel purified and ligated.
  • the ligation reaction was used to transform XLl-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 diagnostic digests were performed ( Figure 7) and one clone was selected and designated pBltSK-TLVH. This clone was used for subsequent DNA manipulations.
  • the final step in the construction of pMOX involved digesting: a) MCOl with Xbal and BglH; b) pBltSK-TLVH with BglH and Sail; and c) pBltSK-PcatCre with Sail and Xbal.
  • MCOl vector, 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 indicates expected restriction digest patterns of the resultant pMOX vector.
  • TcR Tetracyclin resistance
  • the remainder 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 chloramphenicol 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).
  • the loxP-cat gene was restriced with SacI/PstI and ligated into Sacl/Pstl restricted pUT-1 vector.
  • the ligation mixture was transformed into NM522 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 pUTcat) contained, the loxP-cat gene.
  • the next step in the construction of pUX was to clone the lacVH gene into the PstI 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 designated pUX.
  • the tetanus toxoid heavy chain was RE digested with Xhol/Spel and purified in an agarose gel from MCOl-TT. This fragment was ligated into Xhol/Spel RE digested pUX. The ligation mixture 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 TT heavy chain DNA sequences.
  • the tetanus toxoid light chain was RE digested with Sacl Xbal and purified in an agarose gel from MCOl-TT.
  • This fragment was ligated into Sacl/Xbal RE digested pMOX.
  • the ligation mixture was used to transform XLl-blue cells and 12 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-TT and pMOX-TT.
  • pMOX-TT Preparation of pMOX-TT phase.
  • pMOX-TT was transformed into XLl-blue and 250 ⁇ l of an overnight culture was used to inoculate 2YT containing 2% glucose, carbenicillin (carb - 50 ⁇ g/ml)tetracycline (tet - lO ⁇ g/ml) and then rescue with helper phage (VCS- M13).
  • the culture was incubated for 2 hours at 37°C with shaking and thencentrifuged at room temperature at 4500rpm for 15 minutes.
  • the bacterial pellet was resuspended in 50ml of 2YT containing carb 50 ⁇ g/ml, tetlO ⁇ g/ml and kanamycin 70 ⁇ g/ml and incubated overnight at 37°C with shaking.
  • the bacteria were pelleted by centrifugation at ⁇ OOOrpm at room temperature.
  • the phage were precipitated from the supernatant by adding l/5vol 20% PEG6000/2.5M NaCl, mixing and leaving on ice with occasional shaking for 1 hour.
  • the phage were collected by centrifugation at ⁇ OOOrpm for 20 minutes at 4°C.
  • the pellet was resuspended in 500 ⁇ l of 1% BSA, and bacterial debris was removed by a high speed centrifugation in a microfuge tube and stored at 4°C.
  • Carb 25 ⁇ g/ml was added and the culture incubated for 30 minutes with shaking. Further carb (25 ⁇ g/ml) was added and incubated with shaking for a further 3 hours. An aliquot of cells added and incubated with shaking for a further 3 hours.
  • Chloramphenicol further selects for cells that have recombinants containing the pMUX vector since chloramphenicol 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 pMOX-TT into the culture containing HB2151/pUX-TT indicating that recombination had occurred.
  • Pcatrev (MC17) 5'..GGG AAG CTT CCC fATA ACT TCG TAT AGC ATA CAT TAT ACG AAG TTA T1TC GAT AAC TCA AAA AAT ACG CC..3'

Abstract

La présente invention concerne un procédé de production d'un phage vecteur de présentation qui consiste à provoquer ou à permettre la recombinaison entre (i) un premier vecteur qui inclut une séquence codant une première chaîne polypeptidique d'un élément spécifique d'une paire de liaison, et (ii) un second phage vecteur qui inclut une séquence codant une cre recombinase fonctionnellement liée à une séquence témoin qui permet l'expression de la Cre recombinase et une séquence codant une seconde chaîne polypeptidique d'un élément spécifique d'une paire de liaison, l'événement de recombinaison engendrant un phage vecteur de recombinaison dans lequel l'expression de la Cre recombinase est sensiblement inhibée. La présente invention concerne également un phage vecteur qui inclut une séquence nucléotidique codant une séquence promotrice, un premier site de recombinaison, en aval et à proximité de la séquence promotrice, et un cadre de lecture ouvert pour cre recombinase, située en aval et à proximité du site de recombinaison, de telle sorte que le promoteur dirige l'expression de la Cre recombinase.
PCT/AU1996/000555 1995-09-05 1996-09-05 Procede de production de phages vecteurs de presentation WO1997009436A1 (fr)

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JP9510704A JPH10511001A (ja) 1995-09-05 1996-09-05 ファージ提示ベクターの製造法
AU67823/96A AU6782396A (en) 1995-09-05 1996-09-05 Method for producing phage display vectors
EP96928283A EP0850309A1 (fr) 1995-09-05 1996-09-05 Procede de production de phages vecteurs de presentation

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AUPN5239 1995-09-05
AUPN5239A AUPN523995A0 (en) 1995-09-05 1995-09-05 Method for producing phage display vectors

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JP (1) JPH10511001A (fr)
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CA (1) CA2231045A1 (fr)
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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999025821A1 (fr) * 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. Compositions et procedes de modification genetique de plantes
US6143557A (en) * 1995-06-07 2000-11-07 Life Technologies, Inc. Recombination cloning using engineered recombination sites
US6171861B1 (en) 1995-06-07 2001-01-09 Life Technologies, Inc. Recombinational cloning using engineered recombination sites
US6262341B1 (en) 1997-11-18 2001-07-17 Pioneer Hi-Bred International, Inc. Method for the integration of foreign DNA into eukaryotic genomes
US6277608B1 (en) 1997-10-24 2001-08-21 Invitrogen Corporation Recombinational cloning using nucleic acids having recombination sites
US6300545B1 (en) 1997-11-18 2001-10-09 Pioneer Hi-Bred International, Inc. Mobilization of viral genomes from T-DNA using site-specific recombination systems
US6410329B1 (en) 1995-09-25 2002-06-25 Novartis Finance Corporation Method for achieving site specific integration of exogenous DNA delivered by non-biological means to plant cells
WO2004001036A1 (fr) * 2002-06-19 2003-12-31 The Regents Of The University Of California Compositions et procedes de selection de cadres de lecture ouverts
US6720140B1 (en) 1995-06-07 2004-04-13 Invitrogen Corporation Recombinational cloning using engineered recombination sites
US6828093B1 (en) 1997-02-28 2004-12-07 Baylor College Of Medicine Rapid subcloning using site-specific recombination
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
US7560622B2 (en) 2000-10-06 2009-07-14 Pioneer Hi-Bred International, Inc. Methods and compositions relating to the generation of partially transgenic organisms
EP2189539A1 (fr) 2008-11-21 2010-05-26 Chimera Biotec GmbH Complexes de conjugués pour une détection d'analytes
US8685893B2 (en) 1998-07-27 2014-04-01 Genentech, Inc. Phage display
US8883988B2 (en) 1999-03-02 2014-11-11 Life Technologies Corporation Compositions for use in recombinational cloning of nucleic acids
US8945884B2 (en) 2000-12-11 2015-02-03 Life Technologies Corporation Methods and compositions for synthesis of nucleic acid molecules using multiplerecognition sites
US9534252B2 (en) 2003-12-01 2017-01-03 Life Technologies Corporation Nucleic acid molecules containing recombination sites and methods of using the same
US20170302728A1 (en) * 2014-05-21 2017-10-19 Nasdaq Technology Ab Efficient and reliable host distribution of totally ordered global state
WO2018124121A1 (fr) 2016-12-26 2018-07-05 Ｊｃｒファーマ株式会社 Nouvel anticorps anti-récepteur de la transferrine humaine capable de pénétrer dans la barrière hémato-encéphalique
WO2018124107A1 (fr) 2016-12-26 2018-07-05 Ｊｃｒファーマ株式会社 Protéine de fusion comprenant du bdnf
WO2019151539A1 (fr) 2018-02-05 2019-08-08 Ｊｃｒファーマ株式会社 Procédé d'administration d'un médicament à un muscle

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CURRENT BIOLOGY, 4, 12, ZOU et al., "Cre-loxP-Mediated Gene Relacement: A Mouse Strain Producing Humanized Antibodies", pp 1099-1103, 1994. *
FEBS LETTERS, 377, DAVIES & REICHMANN, "An Antibody VH Domain With a lox-Cre Site Integrated Into Its Coding Region: Bacterial Recombination Within a Single Polypeptide Chain", pp 92-96, 1995. *
GENE, 176, TSURUSHITA et al., "Phage Display Vectors for In Vivo Recombination of Immunoglobulin Heavy and Light Chain Genes to Make Large Combinatorial Libraries", pp 59-63, 1996, *
NUCLEIC ACIDS RESEARCH, 21, 9, WATERHOUSE et al., "Combinatorial Infection and In Vivo Recombination: a Strategy for Making Large Phage Antibody Repertoires", pp 2265-2266, 1993. *

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US6143557A (en) * 1995-06-07 2000-11-07 Life Technologies, Inc. Recombination cloning using engineered recombination sites
US6171861B1 (en) 1995-06-07 2001-01-09 Life Technologies, Inc. Recombinational cloning using engineered recombination sites
US6270969B1 (en) 1995-06-07 2001-08-07 Invitrogen Corporation Recombinational cloning using engineered recombination sites
US6720140B1 (en) 1995-06-07 2004-04-13 Invitrogen Corporation Recombinational cloning using engineered recombination sites
US6410329B1 (en) 1995-09-25 2002-06-25 Novartis Finance Corporation Method for achieving site specific integration of exogenous DNA delivered by non-biological means to plant cells
US6828093B1 (en) 1997-02-28 2004-12-07 Baylor College Of Medicine Rapid subcloning using site-specific recombination
JP2009136297A (ja) * 1997-10-24 2009-06-25 Life Technologies Corp 組換え部位を有する核酸を使用する組換えクローニング
US6277608B1 (en) 1997-10-24 2001-08-21 Invitrogen Corporation Recombinational cloning using nucleic acids having recombination sites
US7223601B2 (en) 1997-11-18 2007-05-29 Pioneer Hi-Bred International, Inc. Method for the integration of foreign DNA into eukaryotic genomes
US6187994B1 (en) 1997-11-18 2001-02-13 Pioneer Hi-Bred International, Inc. Compositions and methods for genetic modification of plants
US6455315B1 (en) 1997-11-18 2002-09-24 Pioneer Hi-Bred International, Inc. Compositions and methods to stack multiple nucleotide sequences of interest in the genome of a plant
US6458594B1 (en) 1997-11-18 2002-10-01 Pioneer Hi-Bred International, Inc. Compositions and methods for the targeted removal of a nucleotide sequence from the genome of a plant
US6541231B1 (en) 1997-11-18 2003-04-01 Pioneer Hi-Bred International, Inc. Method for the integration of foreign DNA into eukaryotic genomes
US6552248B1 (en) 1997-11-18 2003-04-22 Pioneer Hi-Bred International, Inc. Compositions and methods for locating preferred integration sites within a plant genome
US6573425B1 (en) 1997-11-18 2003-06-03 Pioneer Hi-Bred International, Inc. Compositions and methods to reduce the complexity of transgene integration in the genome of a plant
US6624297B1 (en) 1997-11-18 2003-09-23 Pioneer Hi-Bred International, Inc. Compositions for the genetic modification of plants
US6664108B1 (en) 1997-11-18 2003-12-16 Pioneer Hi-Bred International, Inc. Mobilization of viral genomes from T-DNA using site-specific recombination systems
US9222098B2 (en) 1997-11-18 2015-12-29 Christopher L. Baszczynski Compositions for the targeted insertion of a nucleotide sequence of interest into the genome of a plant
US6300545B1 (en) 1997-11-18 2001-10-09 Pioneer Hi-Bred International, Inc. Mobilization of viral genomes from T-DNA using site-specific recombination systems
US6262341B1 (en) 1997-11-18 2001-07-17 Pioneer Hi-Bred International, Inc. Method for the integration of foreign DNA into eukaryotic genomes
EP1574573A2 (fr) * 1997-11-18 2005-09-14 Pioneer Hi-Bred International, Inc. Compositions et procedes de modification genetique de plantes
EP1574573A3 (fr) * 1997-11-18 2005-10-26 Pioneer Hi-Bred International, Inc. Compositions et procedes de modification genetique de plantes
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
US7179599B2 (en) 1997-11-18 2007-02-20 Pioneer Hi-Bred International, Inc. Mobilization of viral genomes from T-DNA using site-specific recombination systems
WO1999025821A1 (fr) * 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. Compositions et procedes de modification genetique de plantes
US7361508B2 (en) 1997-11-18 2008-04-22 Pioneer Hi-Bred International, Inc. Compositions and methods for the targeted insertion of a nucleotide sequence of interest into the genome of a plant
US7364902B2 (en) 1997-11-18 2008-04-29 Pioneer Hi-Bred International, Inc, Mobilization of viral genomes from T-DNA using site-specific recombination systems
US7405079B2 (en) 1997-11-18 2008-07-29 Pioneer Hi-Bred International, Inc. Compositions and methods to reduce the complexity of transgene integration into the genome of a plant
US7462766B2 (en) 1997-11-18 2008-12-09 Pioneer Hi-Bred International, Inc. Compositions comprising non-identical recombination sites
US6331661B1 (en) 1997-11-18 2001-12-18 Pioneer Hi-Bred International, Inc. Method for directional stable transformation of eukaryotic cells
US8735158B2 (en) 1997-11-18 2014-05-27 Pioneer Hi-Bred International, Inc. Compositions and methods for the targeted insertion of a nucleotide sequence of interest into the genome of a plant
US7572634B2 (en) 1997-11-18 2009-08-11 Pioneer Hi-Bred International, Inc. Compositions and methods for locating preferred integration sites within the genome of a plant
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US7820880B2 (en) 1997-11-18 2010-10-26 Pioneer Hi-Bred Int'l. Inc. Compositions and methods to stack multiple nucleotide sequences of interest in the genome of a plant
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US8685893B2 (en) 1998-07-27 2014-04-01 Genentech, Inc. Phage display
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US8945884B2 (en) 2000-12-11 2015-02-03 Life Technologies Corporation Methods and compositions for synthesis of nucleic acid molecules using multiplerecognition sites
WO2004001036A1 (fr) * 2002-06-19 2003-12-31 The Regents Of The University Of California Compositions et procedes de selection de cadres de lecture ouverts
US9534252B2 (en) 2003-12-01 2017-01-03 Life Technologies Corporation Nucleic acid molecules containing recombination sites and methods of using the same
EP2189539A1 (fr) 2008-11-21 2010-05-26 Chimera Biotec GmbH Complexes de conjugués pour une détection d'analytes
US8927210B2 (en) 2008-11-21 2015-01-06 Chimera Biotec Gmbh Conjugate complexes for analyte detection
US20170302728A1 (en) * 2014-05-21 2017-10-19 Nasdaq Technology Ab Efficient and reliable host distribution of totally ordered global state
WO2018124121A1 (fr) 2016-12-26 2018-07-05 Ｊｃｒファーマ株式会社 Nouvel anticorps anti-récepteur de la transferrine humaine capable de pénétrer dans la barrière hémato-encéphalique
WO2018124107A1 (fr) 2016-12-26 2018-07-05 Ｊｃｒファーマ株式会社 Protéine de fusion comprenant du bdnf
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Also Published As

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JPH10511001A (ja) 1998-10-27
CA2231045A1 (fr) 1997-03-13
EP0850309A1 (fr) 1998-07-01
AUPN523995A0 (en) 1995-09-28

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