DOI: 10.3724/SP.J.1206.2013.00091

Progress in Biochemistry and Biophysics (生物化学与生物物理进展) 2013/40:12 PP.1209-1219

Comparison of Autotransporter and Ice Nucleation Protein as Carrier Proteins for Antibody Display on The Cell Surface of Escherichia coli

Antibody surface display technology is critical for novel antibody screening and antibody affinity maturation. Currentlymost frequently used display methods are phage display and yeast display. Although Escherichia coli (E. coli) is easily cultured andgenetically manipulated, thus is potentially an excellent display host, the display technology based on E. colihas not been widely used.One of the problems is lack of efficient display of antibodies on the surface of E. coli. Many proteins have been tested as displaycarriers in outer membrane display in E. coli. Display systems based on autotransporter protein (AT) and ice nucleation protein (INP) isthe most extensively studied. Another problem is unstable survival rates of E. coli when antibody is displayed. In this study, wesystematically examined display level, antigen-binding affinity of displayed antibody and survival rate of E. coli using Ag43 β (β domainof Antigen43, an AT protein) and INPNC (fragment of N-terminal and C-terminal of INP) as carrier proteins and T7, lac, araBAD aspromoters for antibody expression. We found that the antigen-binding ability of the Ag43 β based display was superior to that of theINPNC based system. As expected, T7, lac and araBAD promoters drove high, medium and low expressions of antibody. The hostsurvival rate using T7 promoter was extremely low (INPNC: 0.0033%, Ag43 β: 0.02%, the host bearing araBAD promoter had thehighest survival rate (INPNC: 37.80%, Ag43 β: 90.23%), and the lac based system had a survival rate of 2.04% (INPNC) and 13.27%(Ag43 β). Balancing the antigen-binding abilities, antibody expression levels and survival rates, a system using Ag43 β as carrier proteinand lac as promoter is the best choice for antibody display on E. coli.

Key words:cell surface display,autotransporter,INP,scFv,survival rate

ReleaseDate:2015-04-18 09:14:50

[1] Souriau C, Hudson P J. Recombinant antibodies for cancer diagnosis and therapy. Expert Opin Biol Ther, 2003, 3(2): 305-318

[2] Brekke O H, Loset G A. New technologies in therapeutic antibody development. Curr Opin Pharmacol, 2003, 3(5): 544-550

[3] Smith G P. Filamentous fusion phage - novel expression vectors that display cloned antigens on the virion surface. Science, 1985,228(4705): 1315-1317

[4] Boder E T, Wittrup K D. Yeast surface display for directed evolution of protein expression, affinity, and stability. Methods Enzymol,2000, 328: 430-444

[5] Bassi A S, Ding D N, Gloor G B, et al. Expression of single chain antibodies (ScFvs) for c-myc oncoprotein in recombinant Escherichia coli membranes by using the ice-nucleation protein of Pseudomonas syringae. Biotechnol Prog, 2000, 16(4): 557-563

[6] Ramesh B, Sendra V G, Cirino P C, et al. Single-cell characterization of autotransporter-mediated Escherichia coli surface display of disulfide bond-containing proteins. J Biol Chem,2012, 287(46): 38580-38589

[7] Hoet R M, Cohen E H, Kent R B,et al.

[8] Vaughan T J, Williams A J, Pritchard K, et al. Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol, 1996, 14(3): 309-314

[9] Schwartzman S, Fleischmann R, Morgan G J. Do anti-TNF agents have equal efficacy in patients with rheumatoid arthritis?. Arthritis Res Ther, 2004, 6(Suppl 2): S3-S11

[10] Kretzschmar T, Von Ruden T. Antibody discovery: phage display. Curr Opin Biotechnol, 2002, 13(6): 598-602

[11] Daugherty P S, Iverson B L, Georgiou G. Flow cytometric screening of cell-based libraries. J Immunol Methods, 2000, 243 (1-2): 211-227

[12] Sblattero D, Bradbury A.

[13] Harvey B R, Georgiou G, Hayhurst A, et al.

[14] Mazor Y, Van Blarcom T, Mabry R, et al. Isolation of engineered,full-length antibodies from libraries expressed in Escherichia coli.Nat Biotechnol, 2007, 25(5): 563-565

[15] Verhoeven G S, Alexeeva S, Dogterom M, et al. Differential bacterial surface display of peptides by the transmembrane domain of ompA. PLoS One, 2009, 4(8): e6739

[16] Majander K, Korhonen T K, Westerlund-Wikstrom B. Simultaneous display of multiple foreign peptides in the FliD capping and FliC filament proteins of the Escherichia coli flagellum. Appl Environ Microbiol, 2005, 71(8): 4263-4268

[17] Binder U, Matschiner G, Theobald I, et al. High-throughput sorting of an anticalin library via espP-mediated functional display on the Escherichia coil cell surface. J Mol Biol, 2010, 400(4): 783-802

[18] Van Bloois E, Winter R T, Kolmar H, et al. Decorating microbes:surface display of proteins on Escherichia coli. Trends Biotechnol,2011, 29(2): 79-86

[19] Kurland C G, Dong H J. Bacterial growth inhibition by overproduction of protein. Mol Microbiol, 1996, 21(1): 1-4

[20] Dong H J, Nilsson L, Kurland C G. Gratuitous overexpression of genes in Escherichia coli leads to growth-inhibition and ribosome destruction. J Bacteriol, 1995, 177(6): 1497-1504

[21] Terpe K. Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol, 2006, 72 (2):211-222

[22] Dautin N, Bernstein H D. Protein secretion in gram-negative bacteria via the autotransporter pathway. Annu Rev Microbiol, 2007, 61: 89-112

[23] Yim S K, Kim D H, Jung H C, et al. Surface display of heme- and diflavin-containing cytochrome P450 BM3 in Escherichia coli: A whole-cell biocatalyst for oxidation. J Microbiol Biotechnol, 2010,20(4): 712-717

[24] Kang S M, Rhee J K, Kim E J, et al. Bacterial cell surface display for epitope mapping of hepatitis C virus core antigen. FEMS Microbiol Lett, 2003, 226(2): 347-353

[25] Fan L H, Liu N, Yu M R, et al. Cell surface display of carbonic anhydrase on Escherichia coli using ice nucleation protein for CO<>sub2 sequestration. Biotechnol Bioeng, 2011, 108(12): 2853-2864

[26] Studier F W, Moffatt B A. Use of bacteriophage-T7 RNApolymerase to direct selective high-level expression of cloned genes. J Mol Biol, 1986, 189(1): 113-130