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• W2024583841 abstract "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 sc-dsFv 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. 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 sc-dsFv 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) 2The abbreviations used are: scFvsingle chain variable fragmentsc-dsFvsingle chain disulfide-stabilized variable fragmentfXafactor XaVEGFhuman vascular endothelial growth factorPBSphosphate-buffered salineHRPhorseradish peroxidaseELISAenzyme-linked immunosorbent assaycfucolony-forming unitCDRcomplementarity determining region. 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.Yokota T. Milenic D.E. Whitlow M. Schlom J. Cancer Res. 1992; 52: 3402-3408PubMed Google Scholar), making scFv an ideal scaffold for designing tumor-homing molecules carrying therapeutic or imaging agents (for reviews see Refs. 2.Michnick S.W. Sidhu S.S. Nat. Chem. Biol. 2008; 4: 326-329Crossref PubMed Scopus (23) Google Scholar, 3.Wark K.L. Hudson P.J. Adv. Drug Deliv. Rev. 2006; 58: 657-670Crossref PubMed Scopus (71) Google Scholar, 4.Carter P.J. Nat. Rev. Immunol. 2006; 6: 343-357Crossref PubMed Scopus (881) Google Scholar, 5.Hoogenboom H.R. Nat. Biotechnol. 2005; 23: 1105-1116Crossref PubMed Scopus (792) Google Scholar, 6.Holliger P. Hudson P.J. Nat. Biotechnol. 2005; 23: 1126-1136Crossref PubMed Scopus (1453) Google Scholar, 7.Bradbury A.R. Marks J.D. J. Immunol. Methods. 2004; 290: 29-49Crossref PubMed Scopus (236) Google Scholar, 8.Winter G. FEBS Lett. 1998; 430: 92-94Crossref PubMed Scopus (47) Google Scholar). 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.Bird R.E. Hardman K.D. Jacobson J.W. Johnson S. Kaufman B.M. Lee S.M. Lee T. Pope S.H. Riordan G.S. Whitlow M. Science. 1988; 242: 423-426Crossref PubMed Scopus (1306) Google Scholar, 10.Skerra A. Plückthun A. Science. 1988; 240: 1038-1041Crossref PubMed Scopus (826) Google Scholar, 11.Huston J.S. Levinson D. Mudgett-Hunter M. Tai M.S. Novotný J. Margolies M.N. Ridge R.J. Bruccoleri R.E. Haber E. Crea R. Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 5879-5883Crossref PubMed Scopus (1385) Google Scholar). 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.McCafferty J. Griffiths A.D. Winter G. Chiswell D.J. Nature. 1990; 348: 552-554Crossref PubMed Scopus (1886) Google Scholar, 13.Breitling F. Dübel S. Seehaus T. Klewinghaus I. Little M. Gene. 1991; 104: 147-153Crossref PubMed Scopus (250) Google Scholar); 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.Hawkins R.E. Russell S.J. Winter G. J. Mol. Biol. 1992; 226: 889-896Crossref PubMed Scopus (462) Google Scholar, 15.Marks J.D. Griffiths A.D. Malmqvist M. Clackson T.P. Bye J.M. Winter G. Biotechnology. 1992; 10: 779-783Crossref PubMed Scopus (366) Google Scholar). 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 recombinant scFv could play key roles as protein therapeutics and diagnostics. single chain variable fragment single chain disulfide-stabilized variable fragment factor Xa human vascular endothelial growth factor phosphate-buffered saline horseradish peroxidase enzyme-linked immunosorbent assay colony-forming unit complementarity determining region. One shortcoming of the scFv scaffold that frequently hampers the applications of this powerful technology is the aggregation tendency of the scFv molecules under physiological and storage conditions (for reviews see Refs. 16.Raag R. Whitlow M. FASEB J. 1995; 9: 73-80Crossref PubMed Scopus (132) Google Scholar, 17.Reiter Y. Brinkmann U. Lee B. Pastan I. Nat. Biotechnol. 1996; 14: 1239-1245Crossref PubMed Scopus (141) Google Scholar, 18.Hust M. Jostock T. Menzel C. Voedisch B. Mohr A. Brenneis M. Kirsch M.I. Meier D. Dübel S. BMC Biotechnol. 2007; 7: 14Crossref PubMed Scopus (96) Google Scholar, 19.Wörn A. Plückthun A. J. Mol. Biol. 2001; 305: 989-1010Crossref PubMed Scopus (481) Google Scholar). The aggregation mechanism has much to do with the stability of the two variable domains and the dimeric interface (20.Röthlisberger D. Honegger A. Plückthun A. J. Mol. Biol. 2005; 347: 773-789Crossref PubMed Scopus (230) Google Scholar), any nonoptimal interactions within or between the two variable domains could result in unstable dimeric interface and dissociation of the two variable domains in the scFv. The variable domain dissociation has two molecular consequences as follows. First, the variable domains are less stable in isolation and could unfold. Second, the transiently dissociated variable domains could re-associate with supplementary domains from other scFv polypeptide chains, forming higher order complexes composed of more than one polypeptide chain (16.Raag R. Whitlow M. FASEB J. 1995; 9: 73-80Crossref PubMed Scopus (132) Google Scholar). Both situations lead to insoluble aggregates or meta-stable structures. The instability of the scFv structure also compromises the fidelity in reproducing the antibody gene products on the phage surface. This could cause biases in favor of more stable scFv molecules over the less stable ones, in selecting misfolded structures on phage surfaces but nevertheless binding to the antigen (21.Brinkmann U. Chowdhury P.S. Roscoe D.M. Pastan I. J. Immunol. Methods. 1995; 182: 41-50Crossref PubMed Scopus (33) Google Scholar), and in selecting false-positives due to multimeric scFv conformations that are unique only to the phage display systems. This structural instability has thus impacted negatively on the utilities of scFv, leading to uncertainties to the outcomes of the selected and screened scFv molecules in terms of their potential applications in biomedicine. Although the misfolding tendency of scFv has been potentially problematic, the uncertainties because of the intrinsic stability properties of the scFv in phage display remain uncharacterized. A more robust scFv phage display system without the uncertainties due to scFv misfolding would provide an alternative circumventing the downside of the conventional scFv phage display systems. One way to stabilize the scFv scaffold is to engineer a disulfide bond between the two Fv domains, so that the variable domains can be covalently linked with a disulfide bond. Single chain disulfide-stabilized Fv fragment (sc-dsFv) format has been constructed in a single polypeptide chain, as in scFv, with a disulfide bond linking the two variable domains in the framework region (22.Young N.M. MacKenzie C.R. Narang S.A. Oomen R.P. Baenziger J.E. FEBS Lett. 1995; 377: 135-139Crossref PubMed Scopus (70) Google Scholar, 23.Rajagopal V. Pastan I. Kreitman R.J. Protein Eng. 1997; 10: 1453-1459Crossref PubMed Scopus (30) Google Scholar, 24.Wörn A. Plückthun A. Biochemistry. 1999; 38: 8739-8750Crossref PubMed Scopus (125) Google Scholar). The advantages of the sc-dsFv molecules have been demonstrated with the sc-dsFv expressed in E. coli (22.Young N.M. MacKenzie C.R. Narang S.A. Oomen R.P. Baenziger J.E. FEBS Lett. 1995; 377: 135-139Crossref PubMed Scopus (70) Google Scholar, 23.Rajagopal V. Pastan I. Kreitman R.J. Protein Eng. 1997; 10: 1453-1459Crossref PubMed Scopus (30) Google Scholar, 24.Wörn A. Plückthun A. Biochemistry. 1999; 38: 8739-8750Crossref PubMed Scopus (125) Google Scholar). But the single chain disulfide-stabilized Fv fragments have not been expressed on phage surface or as soluble form secreted by E. coli in the culture medium, mostly due to severely decreased yield because of the introduction of the interface cysteines (19.Wörn A. Plückthun A. J. Mol. Biol. 2001; 305: 989-1010Crossref PubMed Scopus (481) Google Scholar, 22.Young N.M. MacKenzie C.R. Narang S.A. Oomen R.P. Baenziger J.E. FEBS Lett. 1995; 377: 135-139Crossref PubMed Scopus (70) Google Scholar, 24.Wörn A. Plückthun A. Biochemistry. 1999; 38: 8739-8750Crossref PubMed Scopus (125) Google Scholar), and phage-displayed sc-dsFv libraries and their applications have not been established. In this work, an sc-dsFv phage display platform has been developed. 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.Thie H. Schirrmann T. Paschke M. Dubel S. Hust M. Nat. Biotechnol. 2008; 25: 49-54Google Scholar, 26.Clérico E.M. Maki J.L. Gierasch L.M. Biopolymers. 2008; 90: 307-319Crossref PubMed Scopus (17) Google Scholar, 27.Steiner D. Forrer P. Stumpp M.T. Plückthun A. Nat. Biotechnol. 2006; 24: 823-831Crossref PubMed Scopus (161) Google Scholar, 28.Hegde R.S. Bernstein H.D. Trends Biochem. Sci. 2006; 31: 563-571Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 29.Kim S.J. Mitra D. Salerno J.R. Hegde R.S. Dev. Cell. 2002; 2: 207-217Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 30.Martoglio B. Dobberstein B. Trends Cell Biol. 1998; 8: 410-415Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 31.Jestin J.L. Volioti G. Winter G. Res. Microbiol. 2001; 152: 187-191Crossref PubMed Scopus (35) Google Scholar), a critical process for the integration of the displayed protein on the recombinant phage surface (32.Rapoza M.P. Webster R.E. J. Bacteriol. 1993; 175: 1856-1859Crossref PubMed Google Scholar). But the optimal signal sequences for the translocation of the pIII fusion protein were not known. 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.Jestin J.L. Volioti G. Winter G. Res. Microbiol. 2001; 152: 187-191Crossref PubMed Scopus (35) Google Scholar). 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. This finding enabled success in increasing the expression of functional anti-VEGF sc-dsFv on M13 phage surface by up to 2 orders of magnitude compared with the expression efficiency due to the wild-type signal sequence. The interface disulfide bond of the phage-displayed sc-dsFv was largely formed, and the binding affinity against VEGF was substantially enhanced. More importantly, synthetic sc-dsFv libraries were expressed successfully on the M13 phage surface with one of the optimum signal sequences, and a large number of the binding sc-dsFv variants against VEGF were compared with the binding scFv variants to assess the effects of the interdomain disulfide bond on the binding sequence preferences of the scFv or the sc-dsFv molecules. This work demonstrates a new antibody display platform with all the benefits from the scFv format but without the drawbacks due to the instability of the scFv structure. The sequence of the template scFv was derived from G6 anti-VEGF Fab (Protein Data Bank code 2FJG (33.Fuh G. Wu P. Liang W.C. Ultsch M. Lee C.V. Moffat B. Wiesmann C. J. Biol. Chem. 2006; 281: 6625-6631Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar)). The construct starts from amino terminus with a hexa-His tag (His)6GH, followed by the Vκ1 light chain variable domain, and then the VH3 heavy chain variable domain. The two variable domains are connected with a linker polypeptide, (G)4SIEGRS(G)4S (for anti-VEGF scFv(fXa+) construct) or (G)4S(G)4S(G)4S (for anti-VEGF scFv(fXa−) construct), where IEGR is the factor Xa (fXa) cutting site. A knowledge-based computer program is available in scanning sequences for tentative fXa cleavage sites in a protein sequence (34.Chen C.T. Yang E.W. Hsu H.J. Sun Y.K. Hsu W.L. Yang A.S. Bioinformatics. 2008; 24: 2691-2697Crossref PubMed Scopus (17) Google Scholar); any possible cleavage sites other than the engineered site could interfere with the fXa-based assay and should be avoided in designing the phage-displayed protein sequence. The sequence of the template scFv is shown in supplemental Fig. 1. The software DNAWorks (helixweb.nih.gov) (35.Hoover D.M. Lubkowski J. Nucleic Acids Res. 2002; 30: e43Crossref PubMed Scopus (377) Google Scholar) was used to optimize the DNA sequence for optimum expression in E. coli and to design a total of 30 overlapping DNA fragments (45 bases each from Integrated DNA Technologies) encoding the sequence of the template scFv. These oligonucleotide fragments were synthesized and ligated with PCR in three stages. Due to the large number of the DNA fragments, it was successful only when the fragments were ligated separately in three groups (a01–a10, a11–a20, and a21–a30, see supplemental Fig. 1B) in the first stage. Each 50-μl PCR contains 5 μl of 10× PCR buffer, 2 μl of 25 mm MgSO4, 1 μl of 50× dNTP mix, 1 μl of 50× high fidelity KOD hot start polymerase Mix (Novagen), and 0.5 μl from each of the 10 μm DNA fragments in water. After hot start for 5 min at 95 °C, 35 PCR cycles (94 °C for 30 s, 57 °C for 30 s, and 68 °C for 25 s) were carried out for each of the three reactions. In the second stage, 5 μl of each of the PCR product mixtures was mixed with respective end fragments (2 μl, 100 μm) into a 50-μl PCR mixture as described above. The same PCR protocol was applied to each of the three reaction mixtures. 10 μl from each of the reaction mixtures were pooled, and the DNA products were extracted with the gel extraction kit from Qiagen. In the third stage, the extracted DNA products were mixed together with the end fragments, i.e. 1st and the 30th DNA fragment (0.5 μl, 10 μm), in a 50-μl PCR mixture, followed by the PCR procedure as described before. The PCR product was purified from excised gel slabs with the gel extraction kit from Qiagen. The agarose gel electrophoresis of the DNA products from the three PCR stages is shown in supplemental Fig. 2. The sequence of the DNA product was confirmed by DNA sequencing. The purified PCR product was digested with the restriction enzymes NotI and SfiI (New England Biolabs). The digested DNA (788 bases) was purified with agarose gel electrophoresis and the gel extraction kit from Qiagen. The phagemid pCANTAB5E (GE Healthcare), digested with the same restriction enzymes NotI and SfiI, was purified with agarose gel electrophoresis and incubated with calf intestinal alkaline phosphatase (New England Biolabs) (1.5 units/μg of DNA) in buffer at 37 °C for 1 h. The 5′-dephosphorylated linear vector was extracted with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with 100% ethanol, washed with 70% ethanol, and dissolved in pure water. 2 μg of the purified linear vector was mixed with the digested PCR product (vector/insert = 1:2 to 1:5 in molar ratio) in the presence of T4 DNA ligase (New England Biolabs) in buffer overnight at 16 °C. The ligation reaction product was examined with agarose gel electrophoresis, as shown in supplemental Fig. 3. The ligation reaction product was purified with phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. The construct of the pCANTAB5E phagemid encoded the scFv, followed by an E-tag sequence and a TAG amber stop codon amino-terminal to the gene III sequence. The E-tag is a marker for the expression of the scFv. The DNA product, dissolved in pure water, was electroporated into an electrocompetent E. coli strain ER2738 (New England Biolabs). The transformed E. coli was plated on LB-ampicillin agar plate overnight. Single colonies were picked, and the insert in the phagemid was confirmed by DNA sequencing. Recombinant phage particles were rescued from the E. coli culture by super-infecting the E. coli with the helper phage M13KO7 (GE Healthcare), precipitated with polyethylene glycol/NaCl, and resuspended in PBS. Human VEGF-121 (VEGF-A residues 34–135 receptor binding domain) (33.Fuh G. Wu P. Liang W.C. Ultsch M. Lee C.V. Moffat B. Wiesmann C. J. Biol. Chem. 2006; 281: 6625-6631Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) was expressed in E. coli as inclusion body; refolding and purification followed the procedure published recently (36.Chang H.J. Hsu H.J. Chang C.F. Peng H.P. Sun Y.K. Yu H.M. Shih H.C. Song C.Y. Lin Y.T. Chen C.C. Wang C.H. Yang A.S. Structure. 2009; 17: 620-631Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Multiple competition curves with various concentrations of immobilized and free VEGF competing for anti-VEGF scFv binding were determined to estimate the EC50 values (the concentration of free VEGF resulting in 50% reduction in binding of the phage-displayed scFv to immobilized VEGF) at the lowest immobilized VEGF concentration (37.DeLano W.L. Cunningham B.C. Clackson T. Lowman H.B. Phage Display. 1st Ed. Oxford University Press, New York2004: 85-94Google Scholar). Wells in the first column of a Maxisorb (Nunc) 96-well plate were coated with human VEGF-A, 2 μg/100 μl. The following wells of the nth column in the plate were coated with (0.25n−1) × 2 μg/100 μl VEGF. After overnight coating at room temperature, the plate was blocked with 5% skim milk in PBST buffer (phosphate-buffered saline with 0.2% Tween 20) for 3 h. The plate was washed three times with PBST and two times with PBS. Recombinant phage displaying the anti-VEGF scFv (109 cfu/50 μl) was mixed with free VEGF (10 μg/50 μl in PBS) and 5% skim milk (50 μl in PBST) in the wells of the first row of the plate; the following wells of the mth row contained the same recombinant phage and skim milk solution, but the VEGF was diluted 10-fold in series, i.e. (0.1m−1) ×10 μg/50 μl for wells in the mth row. The plate was allowed to equilibrate for 7 h at room temperature, washed with 3× PBST and 2× PBS, added anti-M13 antibody was conjugated with HRP (GE Healthcare) for 1 h, washed again with 3× PBST and 2× PBS, and then developed with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Kirkegaard & Perry Laboratories) for 30 min before adding 1% SDS to stop the reaction. Absorbance at 405 nm was measured, and the data were used for EC50 calculations. Primers encoding the altered residues at light chain positions for L:Q38C, L:G41C, L:A43C, L:F98C, and L:Q100C mutations and heavy chain positions for H:Q39C, H:G42C, H:G44C, H:L45C, and H:Q112C mutations were synthesized (Integrated DNA Technologies). The amino acid numbers followed the numbering published in the Protein Data Bank code 2FJG (33.Fuh G. Wu P. Liang W.C. Ultsch M. Lee C.V. Moffat B. Wiesmann C. J. Biol. Chem. 2006; 281: 6625-6631Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The primers were designed with the annealing temperature in the range 60–65 °C. Pairs of cysteines were introduced in one step to the anti-VEGF scFv sequence in the pCANTAB5E phagemid (see above paragraph for the construction of the template phagemid) following the Kunkel protocol (38.Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4540) Google Scholar). The DNA construct of the gene III signal peptide in the pCANTAB5E is shown in Fig. 3. Four libraries spanning the signal peptide region were designed as shown in the figure to identify the critical regions of the signal sequence. The primer sequences for the library constructions are also shown in Fig. 3. For each of the sequence regions to be randomized in amino acid sequence, TAA stop codons were first inserted in the designated place as shown in Fig. 3. The TAA-containing phagemids were used as the parents for the following library construction with the primers containing the degenerate codons NNK (Fig. 3). The TAA stop codons were designed to ensure that only the phagemids carrying degenerate codons would produce pIII fusion protein for phage surface display. Both the TAA-containing phagemids and the phagemid libraries for phage display were constructed with the oligonucleotide-directed mutagenesis initially proposed by Kunkel (38.Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4540) Google Scholar). In this work, we followed the Sidhu and Weiss protocol (39.Sidhu S.S. Weiss G.A. Clackson T. Lowman H.B. Phage Display. 1st Ed. Oxford University Press, New York2004: 27-42Google Scholar). E. coli strand ER2738 was transformed with the phagemid libraries, and the recombinant phage particles were rescued with helper phage M13KO7, precipitated with polyethylene glycol/NaCl, and resuspended in PBS. More details of the phage library preparation can be found in a previous publication (40.Hsu H.J. Tsai K.C. Sun Y.K. Chang H.J. Huang Y.J. Yu H.M. Lin C.H. Mao S.S. Yang A.S. J. Biol. Chem. 2008; 283: 12343-12353Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The construction of the phage display libraries in the CDR regions followed the same procedure above. Wells in a 96-well Maxisorb microtiter plate were coated with VEGF (1 μg/100 μl PBS per well) overnight at room temperature. The wells were then blocked with 5% skim milk in PBST for 1 h and then washed as described before. 100 μl of resuspended phage library (1013 cfu/ml) was added to each well for 1 h under gentle shaking. The plate was cleared with 16 × 300 μl of PBST and 2 × 300 μl of PBS. Vigorous wash steps were intended to remove phages from the well surface because of nonspecific binding. The bound phages were eluted with 100 μl of 0.1 m HCl/glycine (pH 2.2) per well, followed by neutralization with 20 μl of 2 m Tris-base buffer (pH 9.1). The eluted phages were mixed with 1 ml of E. coli strand ER2738 (A600 nm = 0.6) for 15 min at 37 °C. Infected E. coli was titered, and the recombinant phage particles were rescued and amplified (in 9 ml of 2× YT, ampicillin 100 μg/ml; after 1 h, add helper phage (1010); wait for another hour before adding 50 ml of medium containing kanamycin 50 μg/ml and ampicillin 100 μg/ml) overnight at 37 °C with vigorous shaking. The phage in the supernatant of the culture was titered, precipitated with polyethylene glycol/NaCl, and resuspended in PBS. The phage solution was ready for the next round of selection. Single E. coli colonies harboring the selected phagemid were randomly picked using GENETIX Qpix II colony picker to a 96-well deep well culture plate. Each well contained 1 ml of 2× YT, 100 μg of ampicillin, 20 μg of tetracycline, and helper phage M13KO7 1 × 109 cfu. The culture plates were incubated at 37 °C and shaken vigorously overnight. After centrifuging at 3000 × g for 30 min at 4 °C, 50 μl of supernatant was mixed with 50 μl of 5% skim milk in a corresponding well of a 96-well Maxisorb microtiter plate, for which the wells were coated with 1 μg/100 μl VEGF and blocked with 5% skim milk. The ELISA developing procedure was identical to that described above under “Competitive Phage ELISA for Affinity Measurement of the Phage-displayed Anti-VEGF scFv,” except that the 3,3′,5,5′-tetramethylbenzidine substrate (Kirkegaard & Perry Laboratories) was used, and absorbance at 450 nm was measured. 13 × 10-cm nitrocellulose membrane (PROTRAN) was coated with VEGF (100 μg/30 ml in PBS) overnight at room temperature. The membrane was blocked with 5% skim milk for 20 min and then laid on a PBS-soaked filter paper over the platform in GENETIX Qpix II. The phage solutions in the 96-well culture plate were arrayed on the membrane with 96-pin gridding tool (FP3S100 from V & P Scientific, Inc; each pin holds 100 nl of phage solution) installed on the gridding robot in GENETIX Qpix II. After arraying the phage solutions, the membrane was then immersed in 5% skim milk in PBS buffer for 20 min before washing with PBST. Phage remaining on the membrane was quantified by immersing the membrane in anti-M13 antibody conjugated with HRP (1:3000 in 5% skim milk PBST) for 30 min and then washing with PBST and PBS. The membrane was allowed to react with Western Lighting Chemiluminescence Reagent Plus (PerkinElmer Life Sciences) following the protocol suggested by the manufacturer. The chemiluminescence signal was recorded with a LAS-3000 imaging system (FujiFilm). Phage particles displaying anti-VEGF scFv(fXa+) derived from standard pCANTAB5E phagemid were tested for VEGF binding with competitive phage ELISA. EC50 values (the concentration of free VEGF resulting in 50% reduction in binding of the phage-displayed scFv to immobilized VEGF) derived from the series of curves of competitive ELISA in Fig. 1 indicated that the Kd (dissociation constant) values of anti-VEGF scFv against VEGF is on the order of 10–100 nm. This value was derived as EC50 approached Kd when both the phage-displayed anti-VEGF scFv concentration and the immobilized VEGF concentration approached zero (37.DeLano W.L. Cunningham B.C. Clackson T. Lowman H.B. Phage Display. 1st Ed. Oxford University Press, New York2004: 85-94Google Scholar). But the signal-to-noise ratio decreased as the immobilized VEGF concentration decreased (Fig. 1), rendering large uncertainty in measuring EC50. Hence, the EC50 values measured in Fig. 1 can only be regarded as a crude estimation of the Kd for the anti-VEGF scFv(fXa+) against VEGF. Nevertheless, the competitive phage ELISA against VEGF, along with the negative control experiment (null control phage without displaying scFv did not show any binding to VEGF, as shown in Fig. 2), confirmed the expression and binding of the phage-displayed anti-VEGF scFv(fXa+) against VEGF with reasonable binding affinity for the following experiments.FIGURE 2Expression level and binding affinity of anti-VEGF sc-dsFv(fXa+) on M13 phage surface or as free soluble protein secreted in the culture medium with the pCANTAB5E phagemid in E. coli strain ER2738. The signal sequence of the pCANTAB5E phagemid is shown in Fig. 3. The x axis shows various single chain antibody variable domain fragment constructs in the pCANTAB5E phagemid. The details" @default.
• W2024583841 created "2016-06-24" @default.
• W2024583841 creator A5003939547 @default.
• W2024583841 creator A5014393887 @default.
• W2024583841 creator A5015938819 @default.
• W2024583841 creator A5018143128 @default.
• W2024583841 creator A5022321461 @default.
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• W2024583841 date "2010-03-01" @default.
• W2024583841 modified "2023-09-26" @default.
• W2024583841 title "Engineering Anti-vascular Endothelial Growth Factor Single Chain Disulfide-stabilized Antibody Variable Fragments (sc-dsFv) with Phage-displayed sc-dsFv Libraries" @default.
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