Cloning in a bacteriophage lambda vector for the display of binding proteins on filamentous phage

Hogrefe, Holly H.; Amberg, Jeff R.; Hay, Beverly N.; Sorge, Joseph A.; Shopes, Bob

doi:10.1016/0378-1119(93)90255-2

Cited by 19 publications

(8 citation statements)

References 13 publications

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“…Amino acid sequences of fusion proteins. Phage expressing the 65-kDa Cry1Ac toxin protein were produced in two different vector systems, the filamentous phage fUSE5 vector of Scott and Smith (34) and the lambda phage SurfZAP phagemid system of Stratagene (17). In both vector systems, we constructed a translational fusion of a gene encoding the active 65-kDa core of Cry1Ac toxin with a sequence encoding a minor filamentous phage coat protein, also known as the attachment protein, cpIII (or pIII).…”

Section: Resultsmentioning

confidence: 99%

“…(ii) SurfZap system. The Stratagene Lambda SurfZAP vector is a 41.5-kb lambda phage vector derived from the LambdaZAP II vector (also from Stratagene), which contains a defective filamentous phage (f1) genome that can be excised as a phagemid (pSurfscript) and packaged into f1 phage particles with the assistance of VCSM13 helper phage (17). A translational fusion of a cry1Ac gene with amino acids 198 to 406 of an f1 phage cpIII gene in the SurfZAP vector allows phage display of Cry1Ac protein on filamentous phage and was constructed as follows.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Phage Display of a Biologically Active Bacillus thuringiensis Toxin

Kasman

Lukowiak

Garczynski

et al. 1998

Appl Environ Microbiol

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Activated forms of Bacillus thuringiensisinsecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensistoxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Phage Display of a Biologically Active Bacillus thuringiensis Toxin

Kasman

Lukowiak

Garczynski

et al. 1998

Appl Environ Microbiol

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“…Our approach with phagemid vectors and helper phagemediated excision allows us to increase the total number of vectors packaged from about 6 -10 units per phage head. SurfZAP™ (29) and lambdaZLG6 (30) are other examples of phage display systems taking advantage of the cloning efficiency of phage lambda. In these systems, however, only a single phagemid is subjected to in vivo excision.…”

Section: Resultsmentioning

confidence: 99%

Selection and Identification of Dense Granule Antigen GRA3 by Toxoplasma gondii Whole Genome Phage Display

Robben

Hertveldt

Bosmans

et al. 2002

Journal of Biological Chemistry

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Toxoplasma gondii is a ubiquitous, unicellular, eukaryotic parasite with a complex intracellular life cycle capable of invading and chronically infecting a wide variety of vertebrate host species, including man. Although normally opportunistic in healthy adults, it is a lethal pathogen in immunocompromised humans, particularly in AIDS patients. We present the application of a genomic phage display as a tool for the direct identification of antigens with potential value in diagnosis and/or as subunit vaccine components. Using a polycosmid cloning strategy, we constructed a large phagemid display library (>10 9 independent clones) of mixed short genomic restriction fragments (< 500 bp) of T. gondii genomic DNA (80 Mbp genome size) fused to gene III of the filamentous phage M13. Biopanning of the library with monoclonal Toxoplasma antibodies resulted in the isolation and identification of an epitope of GRA3, an antigen located in the dense granules of T. gondii tachyzoites. The reactivity of the phage displaying the GRA3 epitope with the monoclonal antibody was confirmed by an enzyme-linked immunosorbent assay. These results demonstrate the accessibility of midsized eukaryotic genomes to display technology and the feasibility to screen these whole genome display libraries with antibodies for isolating novel antigenic determinants.

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“…(6,7,10,11) These methods effectively limit the size of antibody libraries to 108 clones, which was an acceptable limit prior to the advent of phage display technology, which allows the rapid screening of > 1012 clones. To take advantage of the power of phage display screening, Waterhouse et al (4s) have proposed an in vivo recombination method based on lox-Cre site-specific recombination.…”

Section: Discussionmentioning

confidence: 99%

Construction of phagemid display libraries with PCR-amplified immunoglobulin sequences.

Hogrefe¹,

Shopes²

1994

Genome Research

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The expression of active antibody fragments in Escherichia coli has led to significant advances in the identification of antigen-binding clones using molecular biological techniques. (1-7) When coupled with the recently developed phage display technology, (8'0) Fab fragment libraries containing up to 10 8 unique specificities may be readily constructed and screened. Using the phage display screening technique, clones that bind to a particular antigen may be isolated by enriching (biopanning) libraries of recombinant phagemid that display Fab on the surface as a fusion with the minor coat protein cpIII of M13. (1~ The phagemid contain the light-chain and heavychain genes of the displayed Fab, thereby allowing further characterization and manipulation of antigen-binding activity at the molecular level. (ls-2o) The diversity of the immunoglobulin repertoire may be effectively captured by PCR. Light-and heavy-chain genes are amplified by PCR from reverse-transcribed transcripts of the rearranged immunoglobulin genes. To construct comprehensive Fab libraries, exhaustive sets of PCR primers have been designed by us (1~ this article) and others (3' 17,21-26) that correspond to the amino terminus of the variable regions of known immunoglobulins and the carboxyl terminus of the CH1 domains of the heavy-chain isotypes or the K and k light chains.A number of strategies have been devised to recombine diverse repertoires of light-and heavy-chain sequences randomly into a bicistronic operon expressing Fab. These include sequential cloning, (12) PCR with a linker primer, (17) splice overlap extension, (27) ligating together two restriction-digested vectors, each encoding a single chain (6'7) and, finally, ligating the light-and heavy-chain Fd sequences together at a rare nonpalindromic restriction site. (1~ The latter approach represents an efficient, one-step cloning method that avoids potential unnecessary skewing of the immunoglobulin repertoire. With combinatorial approaches, it has been possible to construct Fab libraries containing up to 10 8 unique clones, the limiting factor being transformation of bacteria by plasmid or by infection. Methodologies for constructing larger libraries on the theoretical scale of 1012 clones are currently under development and may permit isolation of rare specificities with high affinity without the necessity of prior immunization. MATERIALS AND METHODS StrategyWe have developed a simplified, but efficient, method for constructing comprehensive phage display libraries of human Fab fragments (Fig. 1). A schematic of the SurfZAP lambda vector (Stratagene Cloning Systems) (1~ with a Fab gene cassette is shown in Figure 2. In our Fab fragment library constructs, the heavy-chain Fd portion (VH-D-J-CH1 domains) is expressed as a fusion with the M13 minor coat protein cpIII, although light-chain-cpIII fusions have been used successfully by others. (16) The light-chain and Fd-cpIII sequences are transcribed as a polycistronic message from the lacZ promoter.The SurfZAP vector encodes a ribosome-b...

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Cloning in a bacteriophage lambda vector for the display of binding proteins on filamentous phage

Cited by 19 publications

References 13 publications

Phage Display of a Biologically Active Bacillus thuringiensis Toxin

Phage Display of a Biologically Active Bacillus thuringiensis Toxin

Selection and Identification of Dense Granule Antigen GRA3 by Toxoplasma gondii Whole Genome Phage Display

Construction of phagemid display libraries with PCR-amplified immunoglobulin sequences.

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