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High-throughput production of human proteins for crystallization: The SGC experience
Open accessAbstract
Producing purified human proteins with high yield and purity remains a considerable challenge. We describe the methods utilized in the Structural Genomics Consortium (SGC) in Oxford, resulting in successful purification of 48% of human proteins attempted; of those, the structures of approximately 40% were solved by X-ray crystallography. The main driver has been the parallel processing of multiple (typically 9-20) truncated constructs of each target; modest diversity in vectors and host systems; and standardized purification procedures. We provide method details as well as data on the properties of the constructs leading to crystallized proteins and the impact of methodological variants. These can be used to formulate guidelines for initial approaches to expression of new eukaryotic proteins.
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Cited by
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DNA-dependent formation of transcription factor pairs alters their binding specificity
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Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases
Canning, P., Cooper, C. D. O., Krojer, T., Murray, J. W., Pike, A. C. W., Chaikuad, A., Keates, T., Thangaratnarajah, C., Hojzan, V., Marsden, B. D., et al. (2013) · Journal of Biological Chemistry · doi:10.1074/jbc.m112.437996 · Cited by 284
“Human Cul1NTD (Q13616, residues 1–412), Cul3NTD (Q13618, residues 1–390), Cul3NTD N22 (residues 23–390), Cul5NTD (Q93034, residues 1–386), and KBTBD5 (Q2TBA0, residues 314–621) were subcloned into the vector pNIC-CTHF using ligation-independent cloning, as described previously (31).”
Self-citationRecent advances in mammalian protein production
Bandaranayake, A. D., & Almo, S. C. (2014) · FEBS Letters · doi:10.1016/j.febslet.2013.11.035 · Cited by 222
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Morreale, F. E., Kleine, S., Leodolter, J., Junker, S., Hoi, D. M., Ovchinnikov, S., Okun, A., Kley, J., Kurzbauer, R., Junk, L., et al. (2022) · Cell · doi:10.1016/j.cell.2022.05.009 · Cited by 196
Metadata
- Authors
- Savitsky, P., Bray, J., Cooper, C. D. O., Marsden, B. D., Mahajan, P., Burgess-Brown, N. A., & Gileadi, O.
- Venue
- Journal of Structural Biology
- Publisher
- Elsevier BV
- Published
- 2010-10
- Volume
- 172
- Issue
- 1
- Pages
- 3-13
- ISSN
- 1047-8477 (ISSN-L)
- OpenAlex
- W2025410059
- Cited by
- 331
- References
- 0
- FWCI
- 6.95
- Type
- journal article
- Language
- en
- Open access
- hybrid
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- Savitsky, P., Bray, J., Cooper, C. D. O., Marsden, B. D., Mahajan, P., Burgess-Brown, N. A., & Gileadi, O. (2010). High-throughput production of human proteins for crystallization: The SGC experience. Journal of Structural Biology. https://doi.org/10.1016/j.jsb.2010.06.008
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