Improving the display of proteins on filamentous phage

Jestin, Jean-Luc; Volioti, Georgia; Winter, Greg

doi:10.1016/s0923-2508(01)01191-3

Cited by 41 publications

(26 citation statements)

References 22 publications

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“…Because the disulfide bonds of the newly synthesized preprotein can only be formed in the oxidizing environment of periplasm, the mechanism for the translocation of the nascent unfolded polypeptide chain from the translation site in the cytoplasm across the periplasm membrane could be a key determinant for the folding and, consequently, for the expression of the displayed protein on the phage surface (25)(26)(27)(28)(29)(30)(31). Alternative sequences in the signal peptide region have been known to modulate the expression level and folding quality of the displayed protein (25,27,31), but difficulty remains in identifying optimum signal sequences in a vast sequence space for some sc-dsFv constructs.…”

Section: Discussionmentioning

confidence: 99%

“…The key discovery is that the signal sequence variants of the sc-dsFv-pIII fusion protein have varying effects in directing the sc-dsFv expression on the recombinant phage surface. The signal sequence has been known to be responsible for the Sec system-dependent translocation of the pIII fusion protein from the translation site in the cytoplasm to the periplasm membrane (25)(26)(27)(28)(29)(30)(31), a critical process for the integration of the displayed protein on the recombinant phage surface (32). But the optimal signal sequences for the translocation of the pIII fusion protein were not known.…”

mentioning

confidence: 99%

“…We used biological combinatorial strategies to diversify the signal sequence with synthetic phage display libraries. The variants in the phage libraries were selected and screened for high expression capabilities (31). The results indicated that the sequence of the signal peptidase cleavage site (c-region) in the signal peptide amino-terminal to the pIII fusion protein is more critically responsible for the expression of the phage-displayed fusion protein.…”

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confidence: 99%

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Engineering Anti-vascular Endothelial Growth Factor Single Chain Disulfide-stabilized Antibody Variable Fragments (sc-dsFv) with Phage-displayed sc-dsFv Libraries

Huang

Chen

et al. 2010

Journal of Biological Chemistry

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Phage display of antibody fragments from natural or synthetic antibody libraries with the single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. However, the nature of the artificial single chain constructs results in unstable proteins expressed on the phage surface or as soluble proteins secreted in the bacterial culture medium. The stability of the variable domain structures can be enhanced with interdomain disulfide bond, but the single chain disulfide-stabilized constructs (sc-dsFv) have yet to be established as a feasible format for bacterial phage display due to diminishing expression levels on the phage surface in known phage display systems. In this work, biological combinatorial searches were used to establish that the c-region of the signal sequence is critically responsible for effective expression and functional folding of the sc-dsFv on the phage surface. The optimum signal sequences increase the expression of functional sc-dsFv by 2 orders of magnitude compared with wild-type signal sequences, enabling the construction of phage-displayed synthetic antivascular endothelial growth factor sc-dsFv libraries. Comparison of the scFv and scdsFv variants selected from the phage-displayed libraries for vascular endothelial growth factor binding revealed the sequence preference differences resulting from the interdomain disulfide bond. These results underlie a new phage display format for antibody fragments with all the benefits from the scFv format but without the downside due to the instability of the dimeric interface in scFv. Single chain variable fragment (scFv)2 displayed as a fusion protein amino-terminal to the pIII minor capsid protein on the filamentous phage surface is one of the most prominent methods in antibody engineering. The small size of the scFv construct enables superior tissue-penetrating capabilities over the whole IgG or Fab fragment (1), making scFv an ideal scaffold for designing tumor-homing molecules carrying therapeutic or imaging agents (for reviews see Refs. 2-8). scFv is a single polypeptide chain antibody fragment construct encoding a light chain variable domain and a heavy chain variable domain, with a flexible linkage peptide connecting the two domains (9 -11). The recombinant antibody fragment frequently retains antigen-recognizing capability rivaling that of the parent antibody. Moreover, an scFv library, which could contain more than one billion scFv variants, can be propagated with an Escherichia coli vector of bacterial phage origin (12, 13); the recombinant phages displaying the scFv variants can be selected or screened for antigen binding and re-amplified with the host E. coli. The sequences of the selected scFv can be further diversified with various mutagenesis technologies for affinity maturation (14, 15). As such, the scFv scaffold supports a powerful in vitro technology mimicking the production of high affinity antibodies in mammalian immune responses to foreign antigens, and the re...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Engineering Anti-vascular Endothelial Growth Factor Single Chain Disulfide-stabilized Antibody Variable Fragments (sc-dsFv) with Phage-displayed sc-dsFv Libraries

Huang

Chen

et al. 2010

Journal of Biological Chemistry

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“…However, the incorporation of peptideligand sequences isolated by phage display libraries into viral, e.g., adeno-associated virus (AAV), vectors was of variable success [25][26][27][28][29][30][31][32][33] because these sequence motifs were selected only for cell binding and not for cell entry with subsequent gene transfer and their binding potential may have changed in an unpredictable manner when they were incorporated into the virus envelope structure. In addition, sequences isolated by phage display are preselected by expression, assembly, folding, transport and (lack of) post-translational modifications in E. coli and not in an eukaryotic cell environment [34][35][36]. Taking all these limitations of conventional vector targeting strategies into account, peptide display libraries based on the gene therapy vector itself were developed.…”

Section: Introductionmentioning

confidence: 99%

Isolation of targeted AAV2 vectors from novel virus display libraries

Waterkamp

Müller

Ying

et al. 2006

The Journal of Gene Medicine

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Random peptide ligands displayed on viral capsids are emerging tools for selection of targeted gene transfer vectors even without prior knowledge of the potential target cell receptor. We have previously introduced adeno-associated viral (AAV)-displayed peptide libraries that ensure encoding of displayed peptides by the packaged AAV genome. A major limitation of these libraries is their contamination with wild-type (wt) AAV. Here we describe a novel and improved library production system that reliably avoids generation of wt AAV by use of a synthetic cap gene. Selection of targeted AAV vectors from wt-containing and the novel wt-free libraries on cell types with different permissivity for wt AAV2 replication suggested the superiority of the wt-free library. However, from both libraries highly specific peptide sequence motifs were selected which improved transduction of cells with moderate or low permissivity for AAV2 replication. Strong reduction of HeLa cell transduction compared to wt AAV2 and only low level transduction of non-target cells by some selected clones showed that not only the efficiency but also the specificity of gene transfer was improved. In conclusion, our study validates and improves the unique potential of virus display libraries for the development of targeted gene transfer vectors.

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“…The most popular polypeptide display technology, phage display technology, has provided a versatile technology for the discovery and characterization of a variety of protein-protein interactions (6,7). However, bacteriophage display has significant limitations, mainly related to the restrictions imparted by Escherichia coli on the expression, assembly, folding, transport, and posttranslational modifications of the viral protein fusions and their incorporation into viral particles (8). Finally, poor expression of the protein in eukaryotic cells can occur after selection using microorganism display platforms significantly delaying scale-up for therapeutic applications.…”

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confidence: 99%

Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells

Changming¹,

Pike²,

Rinkoski³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery and optimization of high-affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage display that would overcome the protein translation limitations of microorganisms. The use of eukaryotic cells would improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression system for clinical production. In this study, we demonstrate that the replication and polypeptide display characteristics of a eukaryotic retrovirus, avian leukosis virus (ALV), offers a robust, eukaryotic version of bacteriophage display. The binding affinity of a model single-chain Fv antibody was optimized by using ALV display, improving affinity >2,000-fold, from micromolar to picomolar levels. We believe ALV display provides an extension to antibody display on microorganisms and offers virus and cell display platforms in a eukaryotic expression system. ALV display should enable an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to improve promising antibody candidates.antibody engineering | avian leukosis virus | protein display technology | antibody binding affinity | avian leukosis virus polypeptide display

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Improving the display of proteins on filamentous phage

Cited by 41 publications

References 22 publications

Engineering Anti-vascular Endothelial Growth Factor Single Chain Disulfide-stabilized Antibody Variable Fragments (sc-dsFv) with Phage-displayed sc-dsFv Libraries

Engineering Anti-vascular Endothelial Growth Factor Single Chain Disulfide-stabilized Antibody Variable Fragments (sc-dsFv) with Phage-displayed sc-dsFv Libraries

Isolation of targeted AAV2 vectors from novel virus display libraries

Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells

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