A High Through-put Platform for Recombinant Antibodies to Folded Proteins

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 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 PubMed Scopus Google Scholar, J. A. S. E. M. J. D. PubMed Scopus Google Scholar). generation of recombinant antibodies provide a renewable of cloned and highly validated antibody and a permanent A. A. antibodies in PubMed Scopus Google Scholar, S. D. Bjorling E. E. C.A. J. A. S. R. L. S. M. L. S. H. S. M. Uhlen M. S. C. P. H. A community for the of affinity PubMed Scopus Google Scholar). antibodies a for and to generate and for renewable antibody reagents J. S. A. S. M. W. A. J. J. of phage display to high antibody generation and PubMed Scopus Google Scholar, L. K. H. and for antibody generation phage display PubMed Scopus Google Scholar, K. S. Nilsson P. K. A. J. a of highly antibodies to human domains phage PubMed Scopus Google have the to and for antigen antibody and K. S. A to generate renewable to the human PubMed Scopus Google Scholar). of for renewable antibody reagents are proteins in chromatin biology transcription factors and epigenetic antigens. to Human Protein Atlas are available antibodies to of the estimated human A of human transcription expression and PubMed Scopus Google and are recombinant sources the of validated recombinant antibodies to TF and their is a have they multiple of to binding of proteins to PubMed Scopus Google Scholar, of transcription for PubMed Scopus Google and thus to the to generate renewable antibody reagents to of proteins antibodies be an for in of their expression in the and their binding and for antigen and antibody are for a to generate renewable antibodies to chromatin antibody generation phage display is not animal where of the target is to the of the in the to and antigen and in an animal S. S. J. the of in display PubMed Scopus Google Scholar). These are but we believe that the in can their of the to with less antigen and we an industrialized platform and for high affinity renewable antibodies that is for and epigenetic chromatin domains of were in multiple expression to a and antigen a for antibody We a highly and phage H. W. A. A. S. R. is not for antigen PubMed Scopus Google Scholar). were to and multiple Fabs of high and expression in E. coli for each of 346 human domain and 211 epigenetic Remarkably, immunofluorescence with multiple antibodies TF in six cell lines that about of human transcription factors predominantly in the but the is These the of translocation in TF biology. describe the and for a platform that we believe will the of high and antibody reagents to These cloned antibodies are available to the academic community for research through the to and a pipeline to generate high renewable recombinant antibodies to human transcription factors TF antigens were generated epigenetic antigens generated the and the E. coli phage were for phage phage were for expression of antibody PubMed Scopus Google were phage were in the of to expression of the that for phage and were with for of expression were with and recombinant for transcription domains were R. S. J. R. C. K. K. H. M. M. L. S. D. P. S. S. R. D. H. L. platform of the PubMed Scopus Google Scholar, R. S. J. C. K. J. K. H. J. M. L. L. D. P. S. R. S. D. H. L. of for and PubMed Scopus Google Scholar, A of PubMed Scopus Google and and were E. coli expression to generate for antigen expression in transcription and and E. were and their expression W. a cell PubMed Scopus Google Scholar, C. of PubMed Scopus Google Scholar). expression were to expression antigen the human human transcription domains chromatin antigens antigens, expression were cloned an expression an and and in E. coli a were and for the of predominantly in were in to the and to a binding to were in and to a to and to the were and being in and E. S. in produced antigens were in E. and for to the pipeline P. C. M. P. S. S. the human proteome for affinity expression of domains and in PubMed Scopus Google Scholar, S. P. J. J. S. C. R. J. S. A. A. A. R. C. R. S. J. A. F. L. R. M. D. K. J. F. S. A. S. A. R. L. L. K. M. S. H. R. D. H. M. S. A. S. K. C. Protein and PubMed Scopus Google Scholar). Protein expression of TF expression in E. coli the E. coli based expression of antigens expression E. coli were in with to an of with for were and to with S. R. M. M. A. J. S. A. high antibody Scholar). a we the a an E. coli in that J. J. C. C. S. S. K. J. S. antibodies and antibody produced in a in PubMed Scopus Google an with and of were with cell recombinant and and to for in an and with based and with Protein were with and for to the pipeline J. J. C. C. S. S. K. J. S. antibodies and antibody produced in a in PubMed Scopus Google Scholar, M. A. S. S. antibodies to proteins in PubMed Scopus Google Scholar). the we the antigen for the of display for PubMed Scopus Google Scholar, J. for generation and 2004; PubMed Scopus Google Scholar). generate for were and a with a to the which a with the in the were a which for to S. of PubMed Scopus Google Scholar, M. D. an for PubMed Scopus Google Scholar, D. D. display for of PubMed Scopus Google Scholar). TF domain with a to expression and proteins generated these were for the ability to generate antibodies phage phage were to S. R. M. M. A. J. S. A. high antibody Scholar, M. A. S. S. antibodies to proteins in PubMed Scopus Google with were automated for of target antigens generated to of phage were with target antigens to antigen the with and and the of phage to were for were with for with a in with and the of binding we a and where antigen binding were the the of for the were to of E. coli phage and with were with Protein A the of a Protein A with to for were and to of with for to the to and can a of binding to each were with for the were with the to the of binding based were of antigen binding S. R. M. M. A. J. S. A. high antibody Scholar). phage automated were to E. coli to for to that were were a and phage were for with to with were for the of to target A target antigen to in were in for to antigen and for a antigen with phage and for with the complex to the and for were with a to the of antibody in and for were with with and with a for the of and were analysis the the and the of that the to be a a of and a were to analysis to of the of the automated the generation of where the were and and were for the of and were and the E. coli expression were in with and to an of expression the of the of to and to for which were Fabs were Protein A and for and the stability of the recombinant we in which to of proteins a high platform for display and recombinant PubMed Scopus Google Scholar). in recombinant to in and to a to were an and and to generate where the to the of the we an where target Fabs were to were for with target antigen in the of a and were and with a to analysis in the affinity of we in with antigen to and for to with in a to for to and with antibody and cell were with in which they were to and with with were with in with and with in for the of a of in in were with for with to the of and with a of human antibody were for cell lines with an and and to and a of to facilitate analysis and of multiple of to in to generate a that be a were of a in of with each for of a of and and a the were for to allow to the to and with to are with for in a which the and with were for binding of of is for of antibodies to native proteins J. S. A. S. M. W. A. J. J. of phage display to high antibody generation and PubMed Scopus Google Scholar, K. S. Nilsson P. K. A. J. a of highly antibodies to human domains phage PubMed Scopus Google Scholar). target antigens in binding reagents phage in we that proteins that are in not antibodies of high and proteins do generate high antibodies to with of to binding of proteins to PubMed Scopus Google of transcription for PubMed Scopus Google TF antigens, we of to that had domain based and R. S. J. R. C. K. K. H. M. M. L. S. D. P. S. S. R. D. H. L. platform of the PubMed Scopus Google Scholar, R. S. J. C. K. J. K. H. J. M. L. L. D. P. S. R. S. D. H. L. of for and PubMed Scopus Google Scholar, A of PubMed Scopus Google and thus a good of a TF antigen domains were in E. coli proteins to facilitate antigen a site and to phage to K. S. Nilsson P. K. A. J. a of highly antibodies to human domains phage PubMed Scopus Google Scholar, P. C. M. P. S. S. the human proteome for affinity expression of domains and in PubMed Scopus Google Scholar, S. P. J. J. S. C. R. J. S. A. A. A. R. C. R. S. J. A. F. L. R. M. D. K. J. F. S. A. S. A. R. L. L. K. M. S. H. R. D. H. M. S. A. S. K. C. Protein and PubMed Scopus Google Scholar, M. A. S. S. antibodies to proteins in PubMed Scopus Google Scholar). and of the TF to to the antibody and were a of antigen these were not the antigen expression pipeline of the of antigens and the of We a phage display where the and domain is to the and the is a M. and in a phage display PubMed Scopus Google Scholar, S. R. an for proteins with binding PubMed Scopus Google Scholar). Fabs in the of E. coli through a with that are and in the phage antibody phage display 2004; PubMed Scopus Google Scholar). is derived the a for which antibody are based M. with and in the of PubMed Scopus Google Scholar, of a monoclonal antibody for the of J. PubMed Scopus Google Scholar, C.A. of and in J. PubMed Scopus Google Scholar). is a and that to the in of the six H. W. A. A. S. R. is not for antigen PubMed Scopus Google Scholar). were in a based analysis of in the H. W. A. A. S. R. is not for antigen PubMed Scopus Google Scholar, K. S. D. A. P. M. C. S. generation of antibodies highly PubMed Scopus Google Scholar, A. J. H. M. S. cell of in antibody PubMed Scopus Google Scholar). and in for of target antigens with H. C. C. domains of cell PubMed Scopus Google M. A. S. S. antibodies to proteins in PubMed Scopus Google Scholar, J. antibodies.Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, of and Natl. Acad. Sci. U.S.A. PubMed Scopus Google and A. S. antibodies for and of Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). the we a pipeline to antigens and pipeline can antigen through phage and of and E. coli We a and for antigens and antigens are to and antigen is a M. A. S. S. antibodies to proteins in PubMed Scopus Google Scholar, K. S. D. A. P. M. C. S. generation of antibodies highly PubMed Scopus Google Scholar). the native of the antigen and the of antigen that antigens are to C. for the of antigen to the of PubMed Scopus Google Scholar). some cases binding of antigen for but the of the with the and phage were with and phage were and in E. coli for expression that display cloned antigens the PubMed Scopus Google the and and of antigen of antigen and which is highly to the of phage to the the were made to the We to phage that display with Protein A about of have a we the phage to to the of binding the of the each the antigen to with of antigen we that of antigen is for the for a is is for animal We binding the and that to of the for antigen binding the were and for These were in a for and binding a We the for binding of the to antigen to a an antigen that with an phage antibody high a of affinity and We estimated the affinity a binding the were with antigen and to bind to the antigen where and to a is the of binding the of binding were to have they were to have and were antigen show for antigen and and an antigen for less than of (typically were and for are and for and expression of the were and E. coli expression W. a cell PubMed Scopus Google Scholar, C. of PubMed Scopus Google Scholar). were E. coli of antibody PubMed Scopus Google for expression Fabs were Protein A that bind to Protein but the for our to Protein A binding analysis and of the binding site Sci. 1999; PubMed Scopus Google Scholar). the of the Fabs generated pipeline we a subset of validated Fabs for expression and in E. coli of Fabs that more than in the of to that commercial antibody reagents are in provide to Fabs for the stability of the antibodies we the of Fabs H. M. of to that PubMed Scopus Google Scholar). of the Fabs a affinity for Fabs that high to with the high expression E. the and high affinity to the TF antigens have through the pipeline and antigens have produced about 3000 validated antibodies that were a a next of antibody we to the Fabs bind to their TF in a and have that human antibodies that are in high of binding in S. R. L. J. R. P. D. P. A for of antibodies with PubMed Scopus Google Scholar). binding estimated the in the binding of antibody to antigen in the of We a to Fabs to binding Fabs S. J. A. S. of affinity and of antibodies a PubMed Scopus Google Scholar). to the ability of the to bind and TF antigen in the of complex human cell with is to and with antigen for binding in the of a cell in where antigens were to and to bind in the of the cell in to the to binding had to be of each Fabs having more than than the were to have binding to proteins in the in an for antigens with to Fabs each not of the to target we believe for Fabs with a high of being of the antibodies that the and the of are more than than we the is we antibodies TF are and we not of antigens to and validated Fabs a of antigen the TF antigens, domain generated Fabs that antigens with Fabs the domains are the and domains with of and of of in a ability to generate high affinity that the and TF domain antibody for SCAN domains of the domains were of the is these the stability of the domains that antigens with a in the in a that We a subset of Fabs that were generated to SCAN antigens in a analysis to of the Fabs to the SCAN domains of cases the Fabs generated the few cases where the for and the target antigens were and in to of we not a the related We a that the a and a of We that the of Fabs antigen antigen is not the for binding We that the of Fabs antigen the of the antigen to the of the phage to bind proteins D. H. K. with binding phage and high target PubMed Scopus Google Scholar). These to antigen with high of of TF antigens generated Fabs that and We some of be in E. we to high antigen expression to E. coli expression expression and we the ability to Fabs TF antigens in E. in transcription and antigen We these antigens through the automated pipeline and based the of Fabs that in the of validated Fabs for the TF antigens the expression and the in aggregate produced validated antigens were more with E. coli and antigen display and antigens produced antibodies Fabs and produced a We that having multiple antigen the of factors can in in the cell the cell to cytosol to nucleus their PubMed Google Scholar). an of renewable antibodies to to to the of these immunofluorescence We six human cell lines that are to of based for each of 270 which we had multiple We a to Fabs in with an to the multiple for each antibody to generate a a of to were to and TF proteins to have cell and These that the Fabs produced were for their target TF and be to TF of each of these are in the binding of Fabs we that ∼70% of the Fabs predominantly to the cytosol and ∼20% to the nucleus these and cell lines in of the in the nucleus cytosol Moreover, some were in the cell their We our with in the Protein which generated affinity antibodies of Remarkably, we that of the antigens where an antibody show the and in were with antigen and with the antibody that of antibody binding analysis and a in with antibodies in cell in to will be to the of these Fabs in are available to the community for a we with the to generate Fabs to an of chromatin proteins and to the these proteins are that are to and are to in their the for the the of the antigens based domain that be and cases we had to the domain expression the phage display 211 antigens we Fabs that and and E. H. A. H. S. S. E. M. A. P. S. M. M. A. L. R. A. L. M. S. S. of a to antibody and for in PubMed Scopus Google an and Fabs to of the epigenetic antigens for binding their target in that of these antibodies the target for antigens where the antigen in some cases binding were the proteins were not high to to the the the is more than for antigen in the of complex the for antigens and Fabs of for the epigenetic of the that the domains of the of of the which had in the is a for antibodies to the human proteome M. Oksvold P. Fagerberg L. Lundberg E. K. M. M. Kampf C. Wester K. Hober S. Wernerus H. Bjorling L. Ponten F. a Human Protein PubMed Scopus Google Scholar, S. Uhlen M. Human and the of PubMed Scopus Google Scholar). to generate antibody reagents with the Human Protein Atlas a antibodies in (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 PubMed Scopus Google Scholar, M. D. J. S. Nilsson P. S. K. M. S. proteome antibody phage PubMed Scopus Google Scholar). antigens were produced to were the human target predominantly to be highly to the and of domain generated affinity antibodies that with to of human is available through the web site of these antibodies are available through commercial but not for the of our to in the Protein Atlas we in for the Remarkably, of the had the the being a and the a recombinant monoclonal having to of the transcription antibodies can have monoclonal antibodies they can bind thus of with proteins in the antibody and binding are to they are derived antibodies the that they are not renewable and thus and are Moreover, the human antigen can to proteins an animal and thus can of F. and J. PubMed Scopus Google Scholar). antibodies a renewable and in the cloned monoclonal and a the community to generate A. A. antibodies in PubMed Scopus Google Scholar, M. S. for in antibody PubMed Scopus Google Scholar). that can be in with in the animal and can allow for of antibody reagents to bind the native and even of antibody PubMed Scopus Google Scholar, M. J. D. J. K. P. Using phage display to antibodies of PubMed Scopus Google Scholar, of recombinant antibodies to PubMed Google Scholar, J. J. A. to the of in a of PubMed Scopus Google Scholar). Moreover, cloned can be genetic and are made available to to an industrialized platform for the of renewable recombinant antibodies phage display and high antigen We show in multiple high and cell antibodies to 346 transcription factors and 211 epigenetic of high antigens is a key in the proteins do not good monoclonal antibodies to native proteins J. S. A. S. M. W. A. J. J. of phage display to high antibody generation and PubMed Scopus Google Scholar). We expression of we to be domains based is in the community that to in to generate high and for A. J. K. biology PubMed Scopus Google Scholar). we high expression to the antigen in in generation of validated antibodies the antigen produced in these We the in are of antigen stability and the and J. S. A. S. M. W. A. J. J. of phage display to high antibody generation and PubMed Scopus Google Scholar). These expression provide for antibodies to antigens. for antibodies the of the These the of these TF that domains were problematic to and and they were the in validated is a to and domains generated the and that they are less highly proteins with to are less than with and is of with the phage of These which antigens more in to high we have and about 3000 validated antibodies the Fabs have We that the for epigenetic antigens of is the antigens were based more to the and phage and stability to facilitate generation of affinity automated the in to the with with antibody and the to to of antibody of the to in the phage and antigen the automated with the and of phage of high affinity that can be problematic for Moreover, for the and for phage Protein A have We of the to a Fabs show high expression in E. are in Protein A a highly and can provide more stability and expression than for derived J. S. A. S. M. W. A. J. J. of phage display to high antibody generation and PubMed Scopus Google which have multiple that in stability and recombinant Fabs had that high to affinity Moreover, these are to a that a in affinity of and can be in to in E. coli Fabs good for in cell E. H. A. H. S. S. E. M. A. P. S. M. M. A. L. R. A. L. M. S. S. of a to antibody and for in PubMed Scopus Google the and S. R. L. J. R. P. D. P. A for of antibodies with PubMed Scopus Google do not these are for antibodies that with Fabs generated to highly homologous SCAN domains high cases where to highly related having is in that we not the and we affinity the antibodies to be with these recombinant antibodies be are in biology. We have Fabs for their ability to bind 270 in six human cell Remarkably, an of the Fabs their target antigen predominantly in the cytosol and about in the These are to the where a of TF antibodies bind in the we the of in the cytosol and nucleus were the six cell the were in in a for each cell and we believe in in that the cytosol to the nucleus We believe these reagents will be for to TF recombinant antibodies have of highly binding reagents based the high phage for each high stability and high and expression in E. binding with binding in the of complex cell and immunofluorescence binding in analysis of the of the antibodies for we do not high the Fabs were to for about Fabs with a to the TF a subset will in We have not the of these antibodies in experiments of but have and E. H. A. H. S. S. E. M. A. P. S. M. M. A. L. R. A. L. M. S. S. of a to antibody and for in PubMed Scopus Google Scholar). more is for these and their for of the and expression for these Fabs are available to academic with through the and the will be made available they the we have the an platform is in for the generation of renewable antibodies to the We are to the and of the and of for display and and and in the for with Fragment antigen-binding domain antigen display immunofluorescence

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.