Abstract
Proteins naturally expressed in eukaryotic organisms often require host chaperones, binding partners, and posttranslational modifications for correct folding. Ideally the heterologous expression system chosen should be as similar to the natural host as possible. For example, mammalian proteins should be expressed in mammalian expression systems. However, this does not guarantee a protein will be expressed in a sufficient high yield for structural or biochemical studies or antibody generation. Often a screening process is undertaken in which many parameters including truncations, point mutations, investigation of orthologs, fusion to peptide or protein tags at the N- or C-terminus, the coexpression of binding partners, and even culture conditions are varied to identify the optimal expression conditions. This requires multiparallel expression screening in mammalian cells similar to that already described for E. coli expression. Here we describe in detail a multiparallel method to express proteins in mammalian suspension cells by transient transfection in 24-well or 96-well blocks.
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References
Dyson MR, Shadbolt SP, Vincent K, Perera R, McCafferty J (2004) Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression. BMC Biotechnol 4:32
Dyson MR (2010) Selection of soluble protein expression constructs: the experimental determination of protein domain boundaries. Biochem Soc Trans 38:908–913
Dyson MR et al (2008) Identification of soluble protein fragments by gene fragmentation and genetic selection. Nucl Acids Res 36:e51
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 7:515–521
Trowitzsch S, Bieniossek C, Nie Y, Garzoni F, Berger I (2010) New baculovirus expression tools for recombinant protein complex production. J Struct Biol 172:45–54
Bushell KM, Söllner C, Schuster-Boeckler B, Bateman A, Wright GJ (2008) Large-scale screening for novel low-affinity extracellular protein interactions. Genome Res 18:622–630
Gonzalez R et al (2010) Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency. Proc Natl Acad Sci 107:3552–3557
Colwill K, Graslund S (2011) A roadmap to generate renewable protein binders to the human proteome. Nat Methods 8:551–558
Dyson MR et al (2011) Map** protein interactions by combining antibody affinity maturation and mass spectrometry. Anal Biochem 417:25–35
Bradbury ARM, Sidhu S, Dubel S, McCafferty J (2011) Beyond natural antibodies: the power of in vitro display technologies. Nat Biotech 29:245–254
Parthiban K et al (2019) A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing. mAbs 11:884–898
Chapple S, Crofts A, Shadbolt SP, McCafferty J, Dyson MR (2006) Multiplexed expression and screening for recombinant protein production in mammalian cells. BMC Biotechnol 6:49
Tom R, Bisson L, Durocher Y (2008) Transfection of HEK293-EBNA1 cells in suspension with linear PEI for production of recombinant proteins. Cold Spring Harbor Protocols 2008:pdb.prot4977
Vazquez-Lombardi R et al (2018) Transient expression of human antibodies in mammalian cells. Nat Protoc 13:99–117
Link AJ, LaBaer J (2008) Construction of nucleic acid programmable protein arrays (NAPPA) 3: isolating DNA plasmids in a 96-well plate format. Cold Spring Harbor Protocols 2008:pdb.prot5058
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Chapple, S.D., Dyson, M.R. (2021). High-Throughput Expression Screening in Mammalian Suspension Cells. In: Chen, Y.W., Yiu, CP.B. (eds) Structural Genomics. Methods in Molecular Biology, vol 2199. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0892-0_7
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DOI: https://doi.org/10.1007/978-1-0716-0892-0_7
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