A helper phage to improve single-chain antibody presentation in phage display

Rondot, S.; Koch, Joachim; Breitling, F.; Dübel, Stefan

doi:10.1038/83567

Cited by 264 publications

(219 citation statements)

References 22 publications

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“…Although unnatural amino acids have been displayed on WT M13 phage in single peptides (14), such a system was not amenable to directed evolution experiments within these constraints. We therefore turned to phagemid display, specifically multivalent hyperphage phagemid display (15,16), which we felt would fulfill these two criteria for both the canonical and unnatural amino acids.…”

Section: Proteins Containing Unnatural Amino Acids Are Correctly Dispmentioning

confidence: 99%

Protein evolution with an expanded genetic code

Liu

Mack

Tsao

et al. 2008

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

143

132

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We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in V H CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded ''synthetic'' genetic code can confer a selective advantage in the directed evolution of proteins with specific properties. directed evolution ͉ phage display ͉ unnatural amino acids W ith few exceptions, the genetic codes of all known organisms specify only the 20 canonical amino acids for protein synthesis. Yet it is quite possible that the ability to encode additional amino acids and their corresponding chemical functionalities would be evolutionarily advantageous, especially since nature's choice of 20 could have been arbitrarily fixed at the point of transition between communal and Darwinian evolution paradigms and subsequently sustained by the code's inertia (1). Furthermore, in the limited scope of laboratory-directed evolution, which concerns only one or few specific functions over a short time rather than general organismal fitness over thousands of years, one can easily envision a selective advantage conferred by additional amino acids. Recent developments in our laboratory allow us to explore this possibility. Specifically, orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs capable of incorporating various unnatural amino acids into proteins in response to unique nonsense and frameshift codons have been added to the translational machinery of Escherichia coli (2). These E. coli (X-E. coli) can now be used for evolution of protein function wherein 21 building blocks, rather than the common 20, are available.Several unnatural amino acids were initially chosen, on the basis of their unique chemistries, for use in our system. For example, X-E. coli genetically encoding the bidentate metalchelating amino acid bipyridyl-alanine (3) are well-suited for the evolution of redox and hydrolytic catalysts, as metal ion binding would not require preorganized primary and secondary ligand shells. Similarly, X-E. coli encoding the reactive 4-boronophenylalanine (4) are well-suited for evolution of receptors specific for glycoproteins or serine protease inhibitors, because the boronate group can form covalent complexes with diols or reactive serine residues. In addition, X-E. coli genetically encoding otherwise posttranslationally modified amino acids, such as sulfotyrosine (5), can be used for evolution of properties that exploit the unique chemical characterist...

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Section: Proteins Containing Unnatural Amino Acids Are Correctly Dispmentioning

confidence: 99%

Protein evolution with an expanded genetic code

Liu

Mack

Tsao

et al. 2008

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

143

132

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“…First, repeat series of biopanning can be performed (Edwards et al, 2003). Second, antibody fragments can be displayed with increased efficiency on virions Rondot et al, 2001). Third, increased numbers of biopanned clones can be tested by robotic high throughput screening (de Wildt et al, 2000;Konthur et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

On the influence of vector design on antibody phage display

Soltes

Hust

et al. 2007

Journal of Biotechnology

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Phage display technology is an established technology particularly useful for the generation of monoclonal antibodies (mAbs). The isolation of phagemid-encoded mAb fragments depends on several features of a phage preparation. The aims of this study were to optimize phage display vectors, and to ascertain if different virion features can be optimized independently of each other.Comparisons were made between phagemid virions assembled by g3p-deficient helper phage, Hyperphage, Ex-phage or Phaberge, or corresponding g3p-sufficient helper phage, M13K07. All g3p-deficient helper phage provided a similar level of antibody display, significantly higher than that of M13K07. Hyperphage packaged virions at least 100-fold more efficiently than did Ex-phage or Phaberge. Phaberge's packaging efficiency improved by using a SupE strain. Different phagemids were also compared. Removal of a 56 base pair fragment from the promoter region resulted in increased display level and increased virion production. This critical fragment encodes a lacZ'-like peptide and is also present in other commonly used phagemids.Increasing display level did not show statistical correlation with phage production, phage infectivity or bacterial growth rate. However, phage production was positively correlated to phage infectivity. In summary, this study demonstrates simultaneously optimization of multiple and independent features of importance for phage selection.

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“…11 These same authors found that strains lacking WaaL (e.g., E. coli CLM24) exhibited more efficient N-glycosylation of a native C. jejuni glycoprotein by PglB. Another improvement would be to utilize helper phage that lack a copy of the native g3p protein, so-called hyperphage, 32 thereby permitting more efficient incorporation of the engineered MBP DQNAT -g3p fusion into phage particles. Alternatively, the ''helperless'' phage system developed by Bradbury and coworkers could offer a similar improvement.…”

Section: Discussionmentioning

confidence: 99%

A filamentous phage display system for N‐linked glycoproteins

et al. 2010

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We have developed a filamentous phage display system for the detection of asparaginelinked glycoproteins in Escherichia coli that carry a plasmid encoding the protein glycosylation locus (pgl) from Campylobacter jejuni. In our assay, fusion of target glycoproteins to the minor phage coat protein g3p results in the display of glycans on phage. The glyco-epitope displayed on phage is the product of biosynthetic enzymes encoded by the C. jejuni pgl pathway and minimally requires three essential factors: a pathway for oligosaccharide biosynthesis, a functional oligosaccharyltransferase, and an acceptor protein with a D/E-X 1 -N-X 2 -S/T motif. Glycosylated phages could be recovered by lectin chromatography with enrichment factors as high as 2 3 10 5 per round of panning and these enriched phages retained their infectivity after panning. Using this assay, we show that desired glyco-phenotypes can be reliably selected by panning phagedisplayed glycoprotein libraries on lectins that are specific for the glycan. For instance, we used our phage selection to identify permissible residues in the 22 position of the bacterial consensus acceptor site sequence. Taken together, our results demonstrate that a genotype-phenotype link can be established between the phage-associated glyco-epitope and the phagemid-encoded genes for any of the three essential components of the glycosylation process. Thus, we anticipate that our phage display system can be used to isolate interesting variants in any step of the glycosylation process, thereby making it an invaluable tool for genetic analysis of protein glycosylation and for glycoengineering in E. coli cells.

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A helper phage to improve single-chain antibody presentation in phage display

Cited by 264 publications

References 22 publications

Protein evolution with an expanded genetic code

Protein evolution with an expanded genetic code

On the influence of vector design on antibody phage display

A filamentous phage display system for N‐linked glycoproteins

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