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A pipeline for the production of antibody fragments for structural studies using transient expression in HEK 293T cells.

Nettleship JE, Ren J, Rahman N, Berrow NS, Hatherley D, Barclay AN, Owens RJ
Protein Expr Purif 62 83-9 (2008)
Cited: 36 times
EuropePMC logo PMID: 18662785

Abstract

We describe a pipeline for the rapid production of recombinant Fabs derived from mouse monoclonal antibodies suitable for use in structural studies. The pipeline is exemplified by the production of three Fabs derived from the monoclonal antibodies OX108 (anti-CD200 receptor), OX117 and OX119 (anti-SIRPgamma). Heavy and light chain variable domains were inserted into separate expression vectors containing resident constant regions using In-Fusion PCR cloning. Following transient co-expression in HEK 293T cells, secreted Fab fragments were purified by metal chelate chromatography and gel filtration using an automated procedure with yields of up to 4mg/L of cell culture. Following crystallization trials, diffracting crystals were obtained for the recombinant Fabs of OX108 and OX117, and their structures solved to 2.3A and 2.4A, respectively.

Articles - 3dif mentioned but not cited (8)

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  6. The structure of a LAIR1-containing human antibody reveals a novel mechanism of antigen recognition. Hsieh FL, Higgins MK. Elife 6 e27311 (2017)
  7. Crystal structure of signal regulatory protein gamma (SIRPγ) in complex with an antibody Fab fragment. Nettleship JE, Ren J, Scott DJ, Rahman N, Hatherley D, Zhao Y, Stuart DI, Barclay AN, Owens RJ. BMC Struct Biol 13 13 (2013)
  8. Production, purification, crystallization and preliminary X-ray diffraction analysis of the HIV-2-neutralizing V3 loop-specific Fab fragment 7C8. Uchtenhagen H, Sourial S, Friemann R, Ehnlund M, Spetz AL, Harris RA, Madhurantakam C, Achour A. Acta Crystallogr Sect F Struct Biol Cryst Commun 65 705-708 (2009)


Reviews citing this publication (3)

  1. Grading the commercial optical biosensor literature-Class of 2008: 'The Mighty Binders'. Rich RL, Myszka DG. J Mol Recognit 23 1-64 (2010)
  2. Expression of recombinant glycoproteins in mammalian cells: towards an integrative approach to structural biology. Aricescu AR, Owens RJ. Curr Opin Struct Biol 23 345-356 (2013)
  3. The "Sticky Patch" Model of Crystallization and Modification of Proteins for Enhanced Crystallizability. Derewenda ZS, Godzik A. Methods Mol Biol 1607 77-115 (2017)

Articles citing this publication (25)

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  2. SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses. Dejnirattisai W, Huo J, Zhou D, Zahradník J, Supasa P, Liu C, Duyvesteyn HME, Ginn HM, Mentzer AJ, Tuekprakhon A, Nutalai R, Wang B, Dijokaite A, Khan S, Avinoam O, Bahar M, Skelly D, Adele S, Johnson SA, Amini A, Ritter TG, Mason C, Dold C, Pan D, Assadi S, Bellass A, Omo-Dare N, Koeckerling D, Flaxman A, Jenkin D, Aley PK, Voysey M, Costa Clemens SA, Naveca FG, Nascimento V, Nascimento F, Fernandes da Costa C, Resende PC, Pauvolid-Correa A, Siqueira MM, Baillie V, Serafin N, Kwatra G, Da Silva K, Madhi SA, Nunes MC, Malik T, Openshaw PJM, Baillie JK, Semple MG, Townsend AR, Huang KA, Tan TK, Carroll MW, Klenerman P, Barnes E, Dunachie SJ, Constantinides B, Webster H, Crook D, Pollard AJ, Lambe T, OPTIC Consortium, ISARIC4C Consortium, Paterson NG, Williams MA, Hall DR, Fry EE, Mongkolsapaya J, Ren J, Schreiber G, Stuart DI, Screaton GR. Cell 185 467-484.e15 (2022)
  3. Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum. Liu C, Ginn HM, Dejnirattisai W, Supasa P, Wang B, Tuekprakhon A, Nutalai R, Zhou D, Mentzer AJ, Zhao Y, Duyvesteyn HME, López-Camacho C, Slon-Campos J, Walter TS, Skelly D, Johnson SA, Ritter TG, Mason C, Costa Clemens SA, Gomes Naveca F, Nascimento V, Nascimento F, Fernandes da Costa C, Resende PC, Pauvolid-Correa A, Siqueira MM, Dold C, Temperton N, Dong T, Pollard AJ, Knight JC, Crook D, Lambe T, Clutterbuck E, Bibi S, Flaxman A, Bittaye M, Belij-Rammerstorfer S, Gilbert SC, Malik T, Carroll MW, Klenerman P, Barnes E, Dunachie SJ, Baillie V, Serafin N, Ditse Z, Da Silva K, Paterson NG, Williams MA, Hall DR, Madhi S, Nunes MC, Goulder P, Fry EE, Mongkolsapaya J, Ren J, Stuart DI, Screaton GR. Cell 184 4220-4236.e13 (2021)
  4. Antibody evasion by the P.1 strain of SARS-CoV-2. Dejnirattisai W, Zhou D, Supasa P, Liu C, Mentzer AJ, Ginn HM, Zhao Y, Duyvesteyn HME, Tuekprakhon A, Nutalai R, Wang B, López-Camacho C, Slon-Campos J, Walter TS, Skelly D, Costa Clemens SA, Naveca FG, Nascimento V, Nascimento F, Fernandes da Costa C, Resende PC, Pauvolid-Correa A, Siqueira MM, Dold C, Levin R, Dong T, Pollard AJ, Knight JC, Crook D, Lambe T, Clutterbuck E, Bibi S, Flaxman A, Bittaye M, Belij-Rammerstorfer S, Gilbert SC, Carroll MW, Klenerman P, Barnes E, Dunachie SJ, Paterson NG, Williams MA, Williams MA, Hall DR, Hulswit RJG, Bowden TA, Fry EE, Mongkolsapaya J, Ren J, Stuart DI, Screaton GR. Cell 184 2939-2954.e9 (2021)
  5. Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera. Supasa P, Zhou D, Dejnirattisai W, Liu C, Mentzer AJ, Ginn HM, Zhao Y, Duyvesteyn HME, Nutalai R, Tuekprakhon A, Wang B, Paesen GC, Slon-Campos J, López-Camacho C, Hallis B, Coombes N, Bewley KR, Charlton S, Walter TS, Barnes E, Dunachie SJ, Skelly D, Lumley SF, Baker N, Shaik I, Humphries HE, Godwin K, Gent N, Sienkiewicz A, Dold C, Levin R, Dong T, Pollard AJ, Knight JC, Klenerman P, Crook D, Lambe T, Clutterbuck E, Bibi S, Flaxman A, Bittaye M, Belij-Rammerstorfer S, Gilbert S, Hall DR, Williams MA, Paterson NG, James W, Carroll MW, Fry EE, Mongkolsapaya J, Ren J, Stuart DI, Screaton GR. Cell 184 2201-2211.e7 (2021)
  6. Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum. Tuekprakhon A, Nutalai R, Dijokaite-Guraliuc A, Zhou D, Ginn HM, Selvaraj M, Liu C, Mentzer AJ, Supasa P, Duyvesteyn HME, Das R, Skelly D, Ritter TG, Amini A, Bibi S, Adele S, Johnson SA, Constantinides B, Webster H, Temperton N, Klenerman P, Barnes E, Dunachie SJ, Crook D, Pollard AJ, Lambe T, Goulder P, Paterson NG, Williams MA, Hall DR, OPTIC Consortium, ISARIC4C Consortium, Fry EE, Huo J, Mongkolsapaya J, Ren J, Stuart DI, Screaton GR. Cell 185 2422-2433.e13 (2022)
  7. The antigenic anatomy of SARS-CoV-2 receptor binding domain. Dejnirattisai W, Zhou D, Ginn HM, Duyvesteyn HME, Supasa P, Case JB, Zhao Y, Walter TS, Mentzer AJ, Liu C, Wang B, Paesen GC, Slon-Campos J, López-Camacho C, Kafai NM, Bailey AL, Chen RE, Ying B, Thompson C, Bolton J, Fyfe A, Gupta S, Tan TK, Gilbert-Jaramillo J, James W, Knight M, Carroll MW, Skelly D, Dold C, Peng Y, Levin R, Dong T, Pollard AJ, Knight JC, Klenerman P, Temperton N, Hall DR, Williams MA, Paterson NG, Bertram FKR, Siebert CA, Clare DK, Howe A, Radecke J, Song Y, Townsend AR, Huang KA, Fry EE, Mongkolsapaya J, Diamond MS, Ren J, Stuart DI, Screaton GR. Cell 184 2183-2200.e22 (2021)
  8. Neutralization of SARS-CoV-2 by Destruction of the Prefusion Spike. Huo J, Zhao Y, Ren J, Zhou D, Duyvesteyn HME, Ginn HM, Carrique L, Malinauskas T, Ruza RR, Shah PNM, Tan TK, Rijal P, Coombes N, Bewley KR, Tree JA, Radecke J, Paterson NG, Supasa P, Mongkolsapaya J, Screaton GR, Carroll M, Townsend A, Fry EE, Owens RJ, Stuart DI. Cell Host Microbe 28 445-454.e6 (2020)
  9. Expression of recombinant antibodies. Frenzel A, Hust M, Schirrmann T. Front Immunol 4 217 (2013)
  10. Recent advances in the production of proteins in insect and mammalian cells for structural biology. Nettleship JE, Assenberg R, Diprose JM, Rahman-Huq N, Owens RJ. J Struct Biol 172 55-65 (2010)
  11. Structure of the discoidin domain receptor 1 extracellular region bound to an inhibitory Fab fragment reveals features important for signaling. Carafoli F, Mayer MC, Shiraishi K, Pecheva MA, Chan LY, Nan R, Leitinger B, Hohenester E. Structure 20 688-697 (2012)
  12. Structure of an antibody in complex with its mucin domain linear epitope that is protective against Ebola virus. Olal D, Kuehne AI, Bale S, Halfmann P, Hashiguchi T, Fusco ML, Lee JE, King LB, Kawaoka Y, Dye JM, Saphire EO. J Virol 86 2809-2816 (2012)
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  14. Potent cross-reactive antibodies following Omicron breakthrough in vaccinees. Nutalai R, Zhou D, Tuekprakhon A, Ginn HM, Supasa P, Liu C, Huo J, Mentzer AJ, Duyvesteyn HME, Dijokaite-Guraliuc A, Skelly D, Ritter TG, Amini A, Bibi S, Adele S, Johnson SA, Constantinides B, Webster H, Temperton N, Klenerman P, Barnes E, Dunachie SJ, Crook D, Pollard AJ, Lambe T, Goulder P, OPTIC consortium, ISARIC4C consortium, Paterson NG, Williams MA, Hall DR, Mongkolsapaya J, Fry EE, Dejnirattisai W, Ren J, Stuart DI, Screaton GR. Cell 185 2116-2131.e18 (2022)
  15. The antibody response to SARS-CoV-2 Beta underscores the antigenic distance to other variants. Liu C, Zhou D, Nutalai R, Duyvesteyn HME, Tuekprakhon A, Ginn HM, Dejnirattisai W, Supasa P, Mentzer AJ, Wang B, Case JB, Zhao Y, Skelly DT, Chen RE, Johnson SA, Ritter TG, Mason C, Malik T, Temperton N, Paterson NG, Williams MA, Hall DR, Clare DK, Howe A, Goulder PJR, Fry EE, Diamond MS, Mongkolsapaya J, Ren J, Stuart DI, Screaton GR. Cell Host Microbe 30 53-68.e12 (2022)
  16. A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization. Ooi A, Wong A, Esau L, Lemtiri-Chlieh F, Gehring C. Front Physiol 7 300 (2016)
  17. Characterization of a potent and highly unusual minimally enhancing antibody directed against dengue virus. Renner M, Flanagan A, Dejnirattisai W, Puttikhunt C, Kasinrerk W, Supasa P, Wongwiwat W, Chawansuntati K, Duangchinda T, Cowper A, Midgley CM, Malasit P, Huiskonen JT, Mongkolsapaya J, Screaton GR, Grimes JM. Nat Immunol 19 1248-1256 (2018)
  18. A delicate balance between antibody evasion and ACE2 affinity for Omicron BA.2.75. Huo J, Dijokaite-Guraliuc A, Liu C, Zhou D, Ginn HM, Das R, Supasa P, Selvaraj M, Nutalai R, Tuekprakhon A, Duyvesteyn HME, Mentzer AJ, Skelly D, Ritter TG, Amini A, Bibi S, Adele S, Johnson SA, Paterson NG, Williams MA, Hall DR, Plowright M, Newman TAH, Hornsby H, de Silva TI, Temperton N, Klenerman P, Barnes E, Dunachie SJ, Pollard AJ, Lambe T, Goulder P, OPTIC consortium, ISARIC4C consortium, Fry EE, Mongkolsapaya J, Ren J, Stuart DI, Screaton GR. Cell Rep 42 111903 (2023)
  19. Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans. Holzer B, Rijal P, McNee A, Paudyal B, Martini V, Clark B, Manjegowda T, Salguero FJ, Bessell E, Schwartz JC, Moffat K, Pedrera M, Graham SP, Noble A, Bonnet-Di Placido M, La Ragione RM, Mwangi W, Beverley P, McCauley JW, Daniels RS, Hammond JA, Townsend AR, Tchilian E. PLoS Pathog 17 e1009330 (2021)
  20. Structural and functional analysis of protective antibodies targeting the threefold plateau of enterovirus 71. Huang KA, Zhou D, Fry EE, Kotecha A, Huang PN, Yang SL, Tsao KC, Huang YC, Lin TY, Ren J, Stuart DI. Nat Commun 11 5253 (2020)
  21. The role of the light chain in the structure and binding activity of two cattle antibodies that neutralize bovine respiratory syncytial virus. Ren J, Nettleship JE, Harris G, Mwangi W, Rhaman N, Grant C, Kotecha A, Fry E, Charleston B, Stuart DI, Hammond J, Owens RJ. Mol Immunol 112 123-130 (2019)
  22. Generation and characterisation of recombinant FMDV antibodies: Applications for advancing diagnostic and laboratory assays. Shimmon G, Kotecha A, Ren J, Asfor AS, Newman J, Berryman S, Cottam EM, Gold S, Tuthill TJ, King DP, Brocchi E, King AMQ, Owens R, Fry EE, Stuart DI, Burman A, Jackson T. PLoS One 13 e0201853 (2018)
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  24. Fc-Mediated Functions of Porcine IgG Subclasses. Paudyal B, Mwangi W, Rijal P, Schwartz JC, Noble A, Shaw A, Sealy JE, Bonnet-Di Placido M, Graham SP, Townsend A, Hammond JA, Tchilian E. Front Immunol 13 903755 (2022)
  25. Rapid escape of new SARS-CoV-2 Omicron variants from BA.2-directed antibody responses. Dijokaite-Guraliuc A, Das R, Zhou D, Ginn HM, Liu C, Duyvesteyn HME, Huo J, Nutalai R, Supasa P, Selvaraj M, de Silva TI, Plowright M, Newman TAH, Hornsby H, Mentzer AJ, Skelly D, Ritter TG, Temperton N, Klenerman P, Barnes E, Dunachie SJ, OPTIC consortium, Roemer C, Peacock TP, Paterson NG, Williams MA, Hall DR, Fry EE, Mongkolsapaya J, Ren J, Stuart DI, Screaton GR. Cell Rep 42 112271 (2023)


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