Multiplexed expression and screening for recombinant protein production in mammalian cells

Springer Science and Business Media LLC - Tập 6 Số 1 - 2006
S.D.J. Chapple1, Anna M Crofts1, S Paul Shadbolt1, John McCafferty1, Michael R. Dyson1
1The Atlas of Protein Expression Project, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK

Tóm tắt

Abstract Background A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner. Results A nested PCR approach was used to clone 21 extracellular domains of mouse receptors as CD4 fusions within a mammalian GATEWAY expression vector system. Following transient transfection, HEK-293E cells grown in 2 ml cultures in 24-deep well blocks showed similar growth kinetics, viability and recombinant protein expression profiles, to those grown in 50 ml shake flask cultures as judged by western blotting. Following optimisation, fluorescent dot blot analysis of transfection supernatants was shown to be a rapid method for analysing protein expression yielding similar results as western blot analysis. Addition of urea enhanced the binding of glycoproteins to a nitrocellulose membrane. A good correlation was observed between the results of a plate based small scale transient transfection dot blot pre-screen and successful purification of proteins expressed at the 50 ml scale. Conclusion The combination of small scale multi-well plate culture and dot blotting described here will allow the multiplex analysis of different mammalian expression experiments enabling a faster identification of high yield expression constructs or conditions prior to large scale protein production. The methods for parallel GATEWAY cloning and expression of multiple constructs in cell culture will also be useful for applications such as the generation of receptor protein microarrays.

Từ khóa


Tài liệu tham khảo

Chen CS, Zhu H: Protein microarrays. Biotechniques. 2006, 40 (4): 423-425, 427 passim.

Letarte M, Voulgaraki D, Hatherley D, Foster-Cuevas M, Saunders N, Barclay AN: Analysis of leukocyte membrane protein interactions using protein microarrays. BMC Biochemistry. 2005, 6 (1): 2-10.1186/1471-2091-6-2.

Warford A, Howat W, McCafferty J: Expression profiling by high-throughput immunohistochemistry. J Immunol Methods. 2004, 290 (1–2): 81-92. 10.1016/j.jim.2004.04.010.

Konthur Z, Hust M, Dubel S: Perspectives for systematic in vitro antibody generation. Gene. 2005, 364: 19-29. 10.1016/j.gene.2005.05.042.

Chandonia JM, Brenner SE: The impact of structural genomics: expectations and outcomes. Science. 2006, 311 (5759): 347-351. 10.1126/science.1121018.

Baldi L, Muller N, Picasso S, Jacquet R, Girard P, Thanh HP, Derow E, Wurm FM: Transient gene expression in suspension HEK-293 cells: application to large-scale protein production. Biotechnol Prog. 2005, 21 (1): 148-153. 10.1021/bp049830x.

Durocher Y, Perret S, Kamen A: High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res. 2002, 30 (2): E9-10.1093/nar/30.2.e9.

Geisse S, Henke M: Large-scale transient transfection of mammalian cells: a newly emerging attractive option for recombinant protein production. J Struct Funct Genomics. 2005, 6 (2–3): 165-170. 10.1007/s10969-005-2826-4.

Meissner P, Pick H, Kulangara A, Chatellard P, Friedrich K, Wurm FM: Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells. Biotechnol Bioeng. 2001, 75 (2): 197-203. 10.1002/bit.1179.

Pham PL, Perret S, Doan HC, Cass B, St-Laurent G, Kamen A, Durocher Y: Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency. Biotechnol Bioeng. 2003, 84 (3): 332-342. 10.1002/bit.10774.

Neuhaus EM, Mashukova A, Zhang W, Barbour J, Hatt H: A specific heat shock protein enhances the expression of mammalian olfactory receptor proteins. Chem Senses. 2006, 31 (5): 445-452. 10.1093/chemse/bjj049.

Sauerwald TM, Figueroa B, Hardwick JM, Oyler GA, Betenbaugh MJ: Combining caspase and mitochondrial dysfunction inhibitors of apoptosis to limit cell death in mammalian cell cultures. Biotechnol Bioeng. 2006, 94 (2): 362-372. 10.1002/bit.20874.

Bahia D, Cheung R, Buchs M, Geisse S, Hunt I: Optimisation of insect cell growth in deep-well blocks: development of a high-throughput insect cell expression screen. Protein Expr Purif. 2005, 39 (1): 61-70. 10.1016/j.pep.2004.08.020.

Jordan M, Köhne C, Wurm FM: Calcium-phosphate mediated DNA transfer into HEK-293 cells in suspension: Control of physicochemical parameters allows transfection in stirred media: Transfection and protein expression in mammalian cells. Cytotechnology. 1998, 26 (1): 39-47. 10.1023/A:1007917318181.

Schlaeger E-J, Christensen K: Transient gene expression in mammalian cells grown in serum-free suspension culture. Cytotechnology. 1999, 30 (1): 71-83. 10.1023/A:1008000327766.

Brown MH, Barclay AN: Expression of immunoglobulin and scavenger receptor superfamily domains as chimeric proteins with domains 3 and 4 of CD4 for ligand analysis. Protein Eng. 1994, 7 (4): 515-521. 10.1093/protein/7.4.515.

Hartley JL, Temple GF, Brasch MA: DNA cloning using in vitro site-specific recombination. Genome Res. 2000, 10 (11): 1788-1795. 10.1101/gr.143000.

Dyson MR, Shadbolt SP, Vincent KJ, Perera RL, McCafferty J: Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression. BMC Biotechnol. 2004, 4: 32-10.1186/1472-6750-4-32.

Gingrich JC, Davis DR, Nguyen Q: Multiplex detection and quantitation of proteins on western blots using fluorescent probes. Biotechniques. 2000, 29 (3): 636-642.

Pace CN, Vajdos F, Fee L, Grimsley G, Gray T: How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995, 4 (11): 2411-2423.

Wurm FM: Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol. 2004, 22 (11): 1393-1398. 10.1038/nbt1026.

Girard P, Jordan M, Tsao M, Wurm FM: Small-scale bioreactor system for process development and optimization. Biochem Eng J. 2001, 7 (2): 117-119. 10.1016/S1369-703X(00)00110-8.

Chambers SP, Austen DA, Fulghum JR, Kim WM: High-throughput screening for soluble recombinant expressed kinases in Escherichia coli and insect cells. Protein Expr Purif. 2004, 36 (1): 40-47. 10.1016/j.pep.2004.03.003.

Davies A, Greene A, Lullau E, Abbott WM: Optimisation and evaluation of a high-throughput mammalian protein expression system. Protein Expr Purif. 2005, 42 (1): 111-121. 10.1016/j.pep.2005.03.012.

Collins JE, Wright CL, Edwards CA, Davis MP, Grinham JA, Cole CG, Goward ME, Aguado B, Mallya M, Mokrab Y, et al: A genome annotation-driven approach to cloning the human ORFeome. Genome Biol. 2004, 5 (10): R84-10.1186/gb-2004-5-10-r84.

Gill SC, von Hippel PH: Calculation of protein extinction coefficients from amino acid sequence data. Analytical Biochemistry. 1989, 182 (2): 319-326. 10.1016/0003-2697(89)90602-7.

Thông tin
Thông tin xuất bản

Springer Science and Business Media LLC

Tập 6 Số 1

Thông tin tác giả