Abstract
Production of high quality protein is an essential step for both structural and functional studies. Throughput has increased in the past decade by the use of streamlined workflows with standard operating procedures and automation. In this chapter, we describe the Oxford Protein Production Facility (OPPF) pipeline for protein production, from conception, through vector construction, to expression and purification. Results from projects run in the OPPF demonstrate the value of using parallel expression screening of intracellular proteins in both E. coli and insect cells. Transient expression in Human Embryonic Kidney (HEK) cells is used exclusively for production of secreted glycoproteins. Protein purification and quality assessment are independent of the expression system and enable sample preparation to be simplified and streamlined.
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References
Yang ZR et al (2005) RONN: the bio-basis function neural network technique applied to the detection of natively disordered regions in proteins. Bioinformatics 21(16):3369–3376
Kelley LA et al (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10(6):845–858
Pajon A et al (2005) Design of a data model for developing laboratory information management and analysis systems for protein production. Proteins 58(2):278–284
Morris C et al (2011) The Protein Information Management System (PiMS): a generic tool for any structural biology research laboratory. Acta Crystallogr D Biol Crystallogr 67(Pt 4):249–260
Berrow NS et al (2007) A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res 35(6):e45
Berrow NS, Alderton D, Owens RJ (2009) The precise engineering of expression vectors using high-throughput In-Fusion PCR cloning. Methods Mol Biol 498:75–90
Bird LE (2011) High throughput construction and small scale expression screening of multi-tag vectors in Escherichia coli. Methods 55(1):29–37
Bird LE et al (2014) Application of In-Fusion cloning for the parallel construction of E. coli expression vectors. Methods Mol Biol 1116:209–234
Zhao Y, Chapman DA, Jones IM (2003) Improving baculovirus recombination. Nucleic Acids Res 31(2):E6–E6
Nettleship JE et al (2010) Recent advances in the production of proteins in insect and mammalian cells for structural biology. J Struct Biol 172(1):55–65
Aricescu AR, Lu W, Jones EY (2006) A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr D Biol Crystallogr 62(Pt 10):1243–1250
Durocher Y, Perret S, Kamen A (2002) High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res 30(2):E9
Nettleship JE et al (2015) Transient expression in HEK 293 cells: an alternative to E. coli for the production of secreted and intracellular mammalian proteins. Methods Mol Biol 1258:209–222
Brown MH, Barclay AN (1994) Expression of immunoglobulin and scavenger receptor superfamily domains as chimeric proteins with domains 3 and 4 of CD4 for ligand analysis. Protein Eng 7(4):515–521
Nettleship JE, Rahman-Huq N, Owens RJ (2009) The production of glycoproteins by transient expression in Mammalian cells. Methods Mol Biol 498:245–263
Chang VT et al (2007) Glycoprotein structural genomics: solving the glycosylation problem. Structure 15(3):267–273
Nettleship JE et al (2008) Methods for protein characterization by mass spectrometry, thermal shift (ThermoFluor) assay, and multiangle or static light scattering. Methods Mol Biol 426:299–318
Nettleship JE et al (2012) Converting monoclonal antibodies into Fab fragments for transient expression in mammalian cells. Methods Mol Biol 801:137–159
Joshi HJ, Gupta R (2015) Eukaryotic glycosylation: online methods for site prediction on protein sequences. In: Lütteke T, Frank M (eds) Glycoinformatics. Springer, New York, pp 127–137
Steentoft C et al (2013) Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology. EMBO J 32(10):1478–1488
Berman HM et al (2000) The protein data bank. Nucleic Acids Res 28(1):235–242
Gasteiger E et al (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana Press, Totowa, NJ, pp 571–607
Petersen TN et al (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8(10):785–786
Moller S, Croning MD, Apweiler R (2001) Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17(7):646–653
The UniProt Consortium (2017) UniProt: the universal protein knowledgebase. Nucleic Acids Res 45(D1):D158–D169
Acknowledgements
The OPPF was funded by the Medical Research Council, UK (grant MR/K018779/1). The authors wish to thank Louise Bird for help with data analysis.
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Nettleship, J.E., Rada, H., Owens, R.J. (2019). Overview of a High-Throughput Pipeline for Streamlining the Production of Recombinant Proteins. In: Vincentelli, R. (eds) High-Throughput Protein Production and Purification. Methods in Molecular Biology, vol 2025. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9624-7_2
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DOI: https://doi.org/10.1007/978-1-4939-9624-7_2
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