A High Through-put Platform for Recombinant Antibodies to Folded Proteins
2015; Elsevier BV; Volume: 14; Issue: 10 Linguagem: Inglês
10.1074/mcp.o115.052209
ISSN1535-9484
AutoresMichael Hornsby, Marcin Paduch, Shane Miersch, Annika Sääf, Tet Matsuguchi, Brian H. Lee, Karolina Wypisniak, Allison K. Doak, Daniel A. King, Svitlana Usatyuk, Kimberly J. Perry, Vince Lu, William D. Thomas, Judy Luke, Jay S. Goodman, Robert J. Hoey, Darson Lai, Carly Griffin, Zhijian Li, Franco J. Vizeacoumar, Debbie Dong, Elliot Campbell, Stephen K. Anderson, Nan Zhong, S. Gräslund, Shohei Koide, Jason Moffat, Sachdev S. Sidhu, Anthony A. Kossiakoff, James A. Wells,
Tópico(s)Protein purification and stability
ResumoAntibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade. Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade. Antibodies are crucial reagents for biological research and therapeutics. However, reproducibility for antibody reagents is a major concern, especially for polyclonals and even monoclonals where genetic drift of hybridoma stocks can be problematic (1.Pasqualini R. Arap W. Hybridoma-free generation of monoclonal antibodies.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 257-259Crossref PubMed Scopus (49) Google Scholar, 2.Harlow E. Lane D. Using antibodies : a laboratory manual. 1999; xiv: 495Google Scholar). Moreover, some have estimated that less than half of the animal derived antibodies bind their cognate native proteins (3.Berglund L. Bjorling E. Oksvold P. Fagerberg L. Asplund A. Szigyarto C.A. Persson A. Ottosson J. Wernerus H. Nilsson P. Lundberg E. Sivertsson A. Navani S. Wester K. Kampf C. Hober S. Ponten F. Uhlen M. A genecentric Human Protein Atlas for expression profiles based on antibodies.Mol. Cell. Proteomics. 2008; 7: 2019-2027Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 4.Bordeaux J. Welsh A. Agarwal S. Killiam E. Baquero M. Hanna J. Anagnostou V. Rimm D. Antibody validation.BioTechniques. 2010; 48: 197-209Crossref PubMed Scopus (447) Google Scholar). The systematic generation of recombinant antibodies would provide a renewable collection of cloned and highly validated antibody genes and a permanent validation database (5.Bradbury A. Pluckthun A. Reproducibility: standardize antibodies used in research.Nature. 2015; 518: 27-29Crossref PubMed Scopus (440) Google Scholar, 6.Gloriam D.E. Orchard S. Bertinetti D. Bjorling E. Bongcam-Rudloff E. Borrebaeck C.A. Bourbeillon J. Bradbury A.R. de Daruvar A. Dubel S. Frank R. Gibson T.J. Gold L. Haslam N. Herberg F.W. Hiltke T. Hoheisel J.D. Kerrien S. Koegl M. Konthur Z. Korn B. Landegren U. Montecchi-Palazzi L. Palcy S. Rodriguez H. Schweinsberg S. Sievert V. Stoevesandt O. Taussig M.J. Ueffing M. Uhlen M. van der Maarel S. Wingren C. Woollard P. Sherman D.J. Hermjakob H. A community standard format for the representation of protein affinity reagents.Mol. Cell. Proteomics. 2010; 9: 1-10Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Recombinant antibodies also afford a biosynthetic tool kit for recombination and gene fusions to generate new sensors and functional modulators. Other efforts for renewable antibody reagents (7.Schofield D.J. Pope A.R. Clementel V. Buckell J. Chapple S. Clarke K.F. Conquer J.S. Crofts A.M. Crowther S.R. Dyson M.R. Flack G. Griffin G.J. Hooks Y. Howat W.J. Kolb-Kokocinski A. Kunze S. Martin C.D. Maslen G.L. Mitchell J.N. O'Sullivan M. Perera R.L. Roake W. Shadbolt S.P. Vincent K.J. Warford A. Wilson W.E. Xie J. Young J.L. McCafferty J. Application of phage display to high throughput antibody generation and characterization.Genome Biol. 2007; 8: R254Crossref PubMed Scopus (168) Google Scholar, 8.Turunen L. Takkinen K. Soderlund H. Pulli T. Automated panning and screening procedure on microplates for antibody generation from phage display libraries.J. Biomol. Screen. 2009; 14: 282-293Crossref PubMed Scopus (48) Google Scholar, 9.Pershad K. Pavlovic J.D. Graslund S. Nilsson P. Colwill K. Karatt-Vellatt A. Schofield D.J. Dyson M.R. Pawson T. Kay B.K. McCafferty J. Generating a panel of highly specific antibodies to 20 human SH2 domains by phage display.Protein Eng. Des. Sel. 2010; 23: 279-288Crossref PubMed Scopus (44) Google Scholar) have highlighted the need to design robotics and high-throughput platforms for antigen production, antibody selections, and characterization (10.Colwill K. Graslund S. A roadmap to generate renewable protein binders to the human proteome.Nat. Methods. 2011; 8: 551-558Crossref PubMed Scopus (206) Google Scholar). One area of need for renewable antibody reagents are proteins involved in chromatin biology including transcription factors (TFs)1 and epigenetic antigens. According to The Human Protein Atlas (HPA; www.proteinatlas.org), there are commercially available antibodies to 362 of the estimated 1550 human TFs (11.Vaquerizas J.M. Kummerfeld S.K. Teichmann S.A. Luscombe N.M. A census of human transcription factors: function, expression and evolution.Nat. Rev. Genet. 2009; 10: 252-263Crossref PubMed Scopus (1078) Google Scholar) and none are from recombinant sources (www.antibodypedia.org). Thus, the absence of validated recombinant antibodies to profile specific TF interactions and their spatial distribution is needed. As a protein class, TFs have been particularly challenging because they contain multiple domains, often intrinsically disordered (12.Spolar R.S. Record Jr., M.T. Coupling of local folding to site-specific binding of proteins to DNA.Science. 1994; 263: 777-784Crossref PubMed Scopus (1372) Google Scholar, 13.Frankel A.D. Kim P.S. Modular structure of transcription factors: implications for gene regulation.Cell. 1991; 65: 717-719Abstract Full Text PDF PubMed Scopus (200) Google Scholar), and thus difficult to express as full-length proteins. Hence the NIH Common Fund funded this broad effort to generate renewable antibody reagents to this class of proteins (1U54HG006436). Such antibodies would be an important resource for biologists interested in understanding trafficking of TFs, their expression patterns in cells at the protein level, and ultimately their binding sites and partners during signaling. Recombinant technologies for antigen production and antibody selections are poised for a large-scale effort to generate renewable antibodies to chromatin remodeling proteins. Recombinant antibody generation by phage display is not dependent on animal immunizations where control of the target protein is relinquished to the animal's immune system. Maintaining control of the protein status in vitro allows the user to customize selection conditions such as buffer, pH, temperature, and competitor proteins. In vitro methods eliminate antigen proteolysis, clearance, and auto-antigen antiselection in an animal setting (14.Bradbury A.R. Sidhu S. Dubel S. McCafferty J. Beyond natural antibodies: the power of in vitro display technologies.Nat. Biotechnol. 2011; 29: 245-254Crossref PubMed Scopus (411) Google Scholar). These bench-scale technologies are well-honed but we believe that by automating the in vitro selection technologies can fully realize their additional advantages of reducing the processing time from months to weeks, with much less antigen and reduced cost. Here we present an industrialized platform (Fig. 1A and 1B) for generating high affinity renewable antibodies at large scale that is exemplified here for TFs and epigenetic factors, 557 total chromatin remodeling targets. Soluble domains of TFs were expressed in multiple high-throughput expression formats to ensure a soluble and intact antigen at a sufficient quantity for antibody selections ( 18 protein domain folds) and 211 different epigenetic proteins. Remarkably, immunofluorescence with multiple antibodies per TF in six different cell lines showed that about two thirds of human transcription factors tested reside predominantly in the cytosol, but the precise distribution is cell-line dependent. These data highlight the importance of translocation in TF biology. Our studies describe the pitfalls and viable solutions for a high-throughput platform that we believe will greatly accelerate the process of producing renewable, high quality, and evolvable antibody reagents to folded proteins. These cloned antibodies are available to the academic community for research purposes through the recombinant-antibodies.org. An NIH funded project was launched to rapidly develop and implement a high-throughput pipeline designed to generate high quality renewable recombinant antibodies to human transcription factors (U54 HG006436). TF antigens reported here were generated by Rutgers University, epigenetic antigens generated by the Structural Genomics Consortium (SCG), and in-house at the RAN facilities. E. coli strain XL1-blue (Stratagene, Santa Clara, CA) or T1 phage resistant cells were used for phage propagation, whereas strains DH10B (Invitrogen, Grand Island, NY), or other T1 phage resistant cells were used for Fab expression C43 (DE3) Pro+ (16.Koerber J.T. Thomsen N.D. Hannigan B.T. Degrado W.F. Wells J.A. Nature-inspired design of motif-specific antibody scaffolds.Nat. Biotechnol. 2013; 31: 916-921Crossref PubMed Scopus (55) Google Scholar) or BL21 (DE3) were used. XL1-blue or T1 phage resistant cells were grown in the presence of 5 μg/ml tetracycline (Tet) to ensure expression of the F' pilus that allowed for phage infection and DH10B cells were grown with 50 μg/ml carbenicillin (Carb) for production of plasmid DNA. Fab expression cells, C43 (DE3) Pro+, were grown with 50 μg/ml Carb and 25 μg/ml kanamycin (Kan). Where biotinylation was required (Antigen or Fab) recombinant BirA co-expressed or added during purification (Avidity, LLC., Aurora, CO). The genes for transcription factor domains were identified by bioinformatics (17.Xiao R. Anderson S. Aramini J. Belote R. Buchwald W.A. Ciccosanti C. Conover K. Everett J.K. Hamilton K. Huang Y.J. Janjua H. Jiang M. Kornhaber G.J. Lee D.Y. Locke J.Y. Ma L.C. Maglaqui M. Mao L. Mitra S. Patel D. Rossi P. Sahdev S. Sharma S. Shastry R. Swapna G.V. Tong S.N. Wang D. Wang H. Zhao L. Montelione G.T. Acton T.B. The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium.J. Struct. Biol. 2010; 172: 21-33Crossref PubMed Scopus (101) Google Scholar, 18.Acton T.B. Xiao R. Anderson S. Aramini J. Buchwald W.A. Ciccosanti C. Conover K. Everett J. Hamilton K. Huang Y.J. Janjua H. Kornhaber G. Lau J. Lee D.Y. Liu G. Maglaqui M. Ma L. Mao L. Patel D. Rossi P. Sahdev S. Shastry R. Swapna G.V. Tang Y. Tong S. Wang D. Wang H. Zhao L. Montelione G.T. Preparation of protein samples for NMR structure, function, and small-molecule screening studies.Methods Enzymol. 2011; 493: 21-60Crossref PubMed Scopus (72) Google Scholar, 19.Harrison S.C. A structural taxonomy of DNA-binding domains.Nature. 1991; 353: 715-719Crossref PubMed Scopus (502) Google Scholar) and synthesized (Life Technologies, Carlsbad, CA), and were provided as E. coli expression constructs. In order to generate constructs for antigen expression by in vitro transcription and translation (IVTT), and E. coli, gene fragments were PCR amplified from transfer plasmids and recombined into their specific expression vectors by SLICE or Ligase Independent Cloning techniques (20.Zhang Y. Werling U. Edelmann W. SLiCE: a novel bacterial cell extract-based DNA cloning method.Nucleic Acids Res. 2012; 40: e55Crossref PubMed Scopus (326) Google Scholar, 21.Aslanidis C. Pde Jong P.J. Ligation-independent cloning of PCR products (LIC-PCR).Nucleic Acids Res. 1990; 18: 6069-6074Crossref PubMed Scopus (934) Google Scholar). All expression plasmids were sequence verified prior to expression studies. The antigen production group at Rutgers University or at the SGC Consortium provided purified human biotinylated human transcription factor domains or chromatin remodeling antigens as N-terminal tagged AviTag fusions. For Rutgers antigens, expression constructs were synthesized (GenScript Inc., Piscataway, NJ), cloned into an expression vector containing an N-terminal AviTag, HisTag, and TEV cleavage site, and expressed in E. coli Tuner (DE3) harboring a pLysSRARE2 helper plasmid (EMD Millipore, Billerica, MA). Cells were harvested, lysed by sonication, and clarified lysate was loaded onto HisTrap HP (GE Healthcare, Piscataway, NJ) columns for purification. If the protein of interest was predominantly found in inclusion bodies, pellets were lysed in solubilization buffer (binding buffer containing 6 m urea), bound to the HisTrap column, and subjected to a slow refolding gradient into binding buffer without urea prior to elution. Pooled elution fractions were in vitro biotinylated (Avidity LLC), concentrated, and applied to a HiLoad 16/600 Superdex 75pg (GE Healthcare, Piscataway, NJ) column to remove unreacted biotin and ATP. Fractions corresponding to the monomeric peak were pooled and concentrated before being snap frozen in liquid N2 and stored at −80 °C until shipment (Campbell, E. Anderson, S. in preparation). SGC produced antigens were expressed in E. coli, purified by Immobilized Metal Affinity Chromatography, and tested for biotinylation prior to entry into the Fab selection pipeline (22.Keates T. Cooper C.D. Savitsky P. Allerston C.K. Phillips C. Hammarstrom M. Daga N. Berridge G. Mahajan P. Burgess-Brown N.A. Muller S. Graslund S. Gileadi O. Expressing the human proteome for affinity proteomics: optimizing expression of soluble protein domains and in vivo biotinylation.N. Biotechnol. 2012; 29: 515-525Crossref PubMed Scopus (23) Google Scholar, 23.Graslund S. Nordlund P. Weigelt J. Hallberg B.M. Bray J. Gileadi O. Knapp S. Oppermann U. Arrowsmith C. Hui R. Ming J. dhe-Paganon S. Park H.W. Savchenko A. Yee A. Edwards A. Vincentelli R. Cambillau C. Kim R. Kim S.H. Rao Z. Shi Y. Terwilliger T.C. Kim C.Y. Hung L.W. Waldo G.S. Peleg Y. Albeck S. Unger T. Dym O. Prilusky J. Sussman J.L. Stevens R.C. Lesley S.A. Wilson I.A. Joachimiak A. Collart F. Dementieva I. Donnelly M.I. Eschenfeldt W.H. Kim Y. Stols L. Wu R. Zhou M. Burley S.K. Emtage J.S. Sauder J.M. Thompson D. Bain K. Luz J. Gheyi T. Zhang F. Atwell S. Almo S.C. Bonanno J.B. Fiser A. Swaminathan S. Studier F.W. Chance M.R. Sali A. Acton T.B. Xiao R. Zhao L. Ma L.C. Hunt J.F. Tong L. Cunningham K. Inouye M. Anderson S. Janjua H. Shastry R. Ho C.K. Wang D. Wang H. Jiang M. Montelione G.T. Stuart D.I. Owens R.J. Daenke S. Schutz A. Heinemann U. Yokoyama S. Bussow K. Gunsalus K.C. Structural Genomics, C., China Structural Genomics, C., Northeast Structural Genomics C. Protein production and purification.Nat. Methods. 2008; 5: 135-146Crossref PubMed Scopus (649) Google Scholar). Protein expression system comparisons consisted of base-line TF expression in E. coli as intracellular GST fusion constructs (His-GST-Avi-TEV-Antigen) driven from the pTac promoter (supplemental Fig. S1C). E. coli based expression of soluble GST-TF fusion antigens was conducted by standard protein expression methods. Briefly E. coli cultures were grown in Terrific Broth with antibiotics to an OD600 of 0.6–0.8 with 1 mm IPTG for 3–6 h at 37°C, then harvested by centrifugation. Cells were resuspended, lysed by sonication, and clarified prior to purification with GST Sepharose (24.Miersch S. Li Z. Hanna R. McLaughlin M.E. Hornsby M. Matsuguchi T. Paduch M. Saaf A. Wells J. Koide S. Kossiakoff A. Sidhu S.S. Scalable high throughput selection from phage-display synthetic antibody libraries.J. Vis. Exp. 2015; 95: 51492Google Scholar). As a second system we expressed the TFs as His-Avi-TEV N-terminal fusion driven from a T7 promoter using an E. coli in vitro transcription-translation system (IVTT) that was kindly provided by Sutro Biosciences (25.Yin G. Garces E.D. Yang J. Zhang J. Tran C. Steiner A.R. Roos C. Bajad S. Hudak S. Penta K. Zawada J. Pollitt S. Murray C.J. Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system.MAbs. 2012; 4: 217-225Crossref PubMed Scopus (99) Google Scholar) (supplemental Fig. S1D). The cell-free reaction mixture contained an ATP regeneration system supplemented with chaperones and redox enzymes. Briefly, 10 μg of IVTT plasmid constructs were combined with 1 ml cell free extract containing recombinant BirA protein and 50 μm biotin and allowed to shake at 650rpm for 2 h in an Eppendorf Thermomixer Expressed and biotinylated protein was purified over NiNTA agarose (Qiagen, Valencia, CA) with PBS based buffer system and eluted with PBS + 400 mm Imidazole. Protein samples were buffer exchanged with Amicon spin concentrators (EMD Millipore), concentrations determined by Bradford BSA assay (Thermo Fisher, Grand Island, NY), and analyzed for purity by SDS-PAGE prior to entering the selection pipeline (25.Yin G. Garces E.D. Yang J. Zhang J. Tran C. Steiner A.R. Roos C. Bajad S. Hudak S. Penta K. Zawada J. Pollitt S. Murray C.J. Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system.MAbs. 2012; 4: 217-225Crossref PubMed Scopus (99) Google Scholar, 26.Paduch M. Koide A. Uysal S. Rizk S.S. Koide S. Kossiakoff A.A. Generating conformation-specific synthetic antibodies to trap proteins in selected functional states.Methods. 2013; 60: 3-14Crossref PubMed Scopus (61) Google Scholar). In the third system, we displayed the antigen for selection on the surface of yeast (27.Boder E.T. Wittrup K.D. Yeast surface display for screening combinatorial polypeptide libraries.Nat. Biotechnol. 1997; 15: 553-557Crossref PubMed Scopus (1282) Google Scholar, 28.Weaver-Feldhaus J.M. Lou J. Coleman J.R. Siegel R.W. Marks J.D. Feldhaus M.J. Yeast mating for combinatorial Fab library generation and surface display.FEBS Lett. 2004; 564: 24-34Crossref PubMed Scopus (83) Google Scholar). To generate constructs for YAD, gene fragments were PCR amplified from gene synthesis transfer plasmids and then recombined into YAD plasmid. YAD constructs utilized a N-terminal Avi-6xHis-Aga2-TEV protease fusion partner with a C-terminal V5-His tag. The YAD strain, EBY100, was engineered to overexpress agglutinin AGA1 under the galactose inducible Gal1–10 promoter, which formed a disulfide bonds with the antigen-Aga2 protein in the extracellular space. Constructs were biotinylated during secretion by a cytoplasmic or ER localized BirA, which allowed for efficient capture to streptavidin magnetic beads (Promega, Madison, WI) (29.Kay B.K. Thai S. Volgina V.V. High-throughput biotinylation of proteins.Methods Mol. Biol. 2009; 498: 185-196Crossref PubMed Scopus (47) Google Scholar, 30.Ackerman M. Levary D. Tobon G. Hackel B. Orcutt K.D. Wittrup K.D. Highly avid magnetic bead capture: an efficient selection method for de novo protein engineering utilizing yeast surface display.Biotechnol. Prog. 2009; 25: 774-783Crossref PubMed Scopus (66) Google Scholar, 31.Rakestraw J.A. Aird D. Aha P.M. Baynes B.M. Lipovsek D. Secretion-and-capture cell-surface display for selection of target-binding proteins.Protein Eng. Des. Sel. 2011; 24: 525-530Crossref PubMed Scopus (46) Google Scholar). The TF domain was expressed with a C-terminal V5 epitope tag to monitor expression level (supplemental Fig. S1E and S4). The proteins generated from these three systems were then tested for the ability to generate antibodies by phage display. All phage selections were done according to previously established protocols (24.Miersch S. Li Z. Hanna R. McLaughlin M.E. Hornsby M. Matsuguchi T. Paduch M. Saaf A. Wells J. Koide S. Kossiakoff A. Sidhu S.S. Scalable high throughput selection from phage-display synthetic antibody libraries.J. Vis. Exp. 2015; 95: 51492Google Scholar, 26.Paduch M. Koide A. Uysal S. Rizk S.S. Koide S. Kossiakoff A.A. Generating conformation-specific synthetic antibodies to trap proteins in selected functional states.Methods. 2013; 60: 3-14Crossref PubMed Scopus (61) Google Scholar) with several modifications as outlined below. Briefly, Fab-phage selections were automated allowing for multi-parallel processing of biotinylated target antigens either generated in-house or obtained from collaborators. Up to four rounds of soluble phage selections were conducted with biotinylated target antigens bound to streptavidin magnetic beads (Promega). The antigen concentration on the bead was systematically decreased with successive selection round as follows: 100 nm Round 1, 50 nm Round 2, 10 nm Rounds 3 and 4. To maximize throughput and reduce the amount of phage library used, up to 8 antigen-streptavidin beads complexes were combined into one selection well for round 1. Antigen-streptavidin beads were incubated with 1 × 1013 Fab-phage particles from either Library E or F for one hour with gentle mixing on a King Fisher Flex magnetic bead separator (Thermo Scientific) followed by three washes in phosphate buffered saline supplemented with 0.05% Tween-20 and 0.2% bovine serum albumin (PBST+BSA). To reduce the deleterious effects of nonspecific binding phage, we employed a "catch and release" strategy, where specific antigen binding Fab-phage were selectively eluted from the magnetic beads by the addition of 2 μg/well TEV Protease for 10 min. Once liberated from the streptavidin beads, Fab-phage were introduced to 5 ml (Selection Round 1) or 100 μl (Selection Rounds 2–4) of exponentially growing E. coli XL1-Blue or T1 phage resistant cells and propagated overnight at 37 °C with shaking. Once propagated, Fab-phage were recovered from culture medium with Protein A magnetic beads (EMD Millipore) on the King Fisher Flex. Briefly, 20–50 μl of a Protein A magnetic bead slurry was incubated with up to 1 ml culture supernatant for 60 min then beads were collected and washed prior to elution of Fab-phage with 100 μm Acetic acid for 10 min. Tris-base pH 11 buffer was added to neutralize the purified Fab-phage prior to further processing. The initial Fab-phage library and amplified eluents can contain a small subpopulation of streptavidin binding Fab-phage, therefore prior to each selection round Fab-phage pools were incubated with 50 μl streptavidin-beads for 10 min then the beads were removed with the King Fisher Flex to deplete the library of any nonspecific binding Fab-phage. Plate based selections were done by immobilization of antigen on protein binding plates as described previously (24.Miersch S. Li Z. Hanna R. McLaughlin M.E. Hornsby M. Matsuguchi T. Paduch M. Saaf A. Wells J. Koide S. Kossiakoff A. Sidhu S.S. Scalable high throughput selection from phage-display synthetic antibody libraries.J. Vis. Exp. 2015; 95: 51492Google Scholar). Purified phage from automated selections (rounds 3 or 4) were used to infect 100ul E. coli XL1-blue cells grown to log phase (OD600 0.6–0.8) for 20 minutes prior to plating on LB +50ug/ml Carb Omnitray (Nunc Thermo Fisher, Grand Island, NY) such that at least 200 individual colonies were grown per plate. Single colonies were then picked by either a K6–2 Colony Picker (KBiorsystems, San Diego, CA) or manually into 96-deep well blocks containing 0.5ml 2xYT +50ug/ml Carb and 1x109 pfu/ml KO7 helper phage (New England Biolabs, Ipswich, MA). Liquid cultures were grown for 16–18 hours at 37°C with shaking at 600–900 RPM prior to further processing. Two 96-well immunoassay plates (Corning, Corning, NY) coated with 50ng/well neutravidin (Thermo Fisher) were prepared for the ELISA validation of Fab-phage binders to one target antigen. A biotinylated target antigen was diluted to 20 nm in PBST + BSA. 50 μl/well were dispensed in two neutravidin plates, then incubated for 20 min. Direct Binding: Fab-phage containing culture supernatant was added to one antigen coated plate and incubated for 15 min. Competition ELISA: In a separate plate 20 nM antigen was mixed with phage containing culture supernatant and incubated for 15 min with shaking. After incubation, the phage-antigen complex was added to the Competition plate and incubated for 15 min. Both plates were then washed 3 times with PBST a BioTek EL406 or similar plate washer to remove unbound Fab-phage (or Fab-phage-antigen) followed by the addition of 50 μl anti-M13 phage-HRP antibody (GE Healthcare Piscataway, NJ; diluted 1:5000 in PBST + BSA) and incubated for 30 minutes. Plates were again washed 3 times with PBST or PBS then developed with 50 μl TMB substrate (KPL Labs, Gaithersburg, MD) and analyzed with a Tecan M1000 or similar plate reader monitoring OD650 for 5 min. Alternatively, signal development was stopped by the addition of 1M Phosphoric acid and OD450 signals were determined. ELISA data analysis was conducted by first plotting the OD650/min (or OD450). The Direct Binding signal was plotted on the Y-axis and the ratio of Direct/Competition signals that was plotted on the X-axis. In order to be considered a passing Fab-phage a competition ratio of 0.005 units was required. Passing Fab-phage were then subjected to DNA sequence analysis to determine uniqueness of the Fab CDR sequences. Analysis of the sequencing results was automated by the generation of several scripts where the sequences flanking CDR's were recognized and both DNA and amino acid translations were returned for the 4 CDR's of interest (LC3, HC1, HC2, and HC3). Duplicate Fab-phage sequences were removed and only unique Fab-phage clones passed into the cloning pipeline. C43 (DE3) Pro+ E. coli containing expression plasmids were grown in Terrific Broth supplemented with 0.5% glycerol, Carb, Kan, Chlor, and 5 μm Biotin to an OD600 of 0.6–0.8 at 37 °C then Fab expression was induced by the addition of 1 mm IPTG. At the time of induction incubation temperature was reduced to 30°C and allowed to shake for 16–18 h at which time cells were harvested by centrifugation. Recombinant Fabs were purified by Protein A chromatography and buffer exchanged into PBS buffer for subsequent storage and validation assays. To assess the stability of the recombinant Fabs, we employed differential scanning fluorimetry (DSF) in which Sypro Orange (Invitrogen) binds to hydrophobic regions of partially or fully denatured proteins giving a high fluorescent signal (32.Koerber J.T. Hornsby M.J. Wells J.A. An improved single-chain fab platform for efficient display and recombinant expression.J. Mol. Biol. 2015; 427: 576-586Crossref PubMed Scopus (31) Google Scholar). DSF was conducted either on Roche LC480 Lightcycler or similar qRT-PCR instrument in either 96- or 384-well formats. Briefly, purified recombinant Fab was diluted to 2 μm in DSF buffer containing Sypro Orange 4x and PBS then subjected to a temperature gradient (0.5°C/30 s) from 50 to 95°C. Data were continuously acquired at an ∼490 nm and ∼575 nm (excitation and emission wavelengths) then processed to generate first derivative curves where the curve peak corresponds to the melting temperature of the Fab. To assess Fab specificity, we utilized an immunoprecipitation method where target Fabs were first bound to streptavidin magnetic beads (Dynabeads M-280, LifeTechnologies, Grand Island, NY). Fab loaded beads were the
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