Abstract
Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determination. At the Structural Genomics Consortium, we opted for the ligation-independent cloning (LIC) method which provides the throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in 96-well format and provide a choice of vectors suitable for expressing proteins in both E. coli and the baculovirus expression vector system (BEVS).
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Acknowledgments
We would like to thank all the SGC scientists (past and present) who contributed toward the development of the method. The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA), Janssen, Merck KGaA, MSD, Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome. The BacMam vector backbone (pHTBV1.1 ) was kindly provided by Professor Frederick Boyce (Massachusetts General Hospital, Cambridge, MA).
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Strain-Damerell, C., Mahajan, P., Fernandez-Cid, A., Gileadi, O., Burgess-Brown, N.A. (2021). Screening and Production of Recombinant Human Proteins: Ligation-Independent Cloning. 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_3
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DOI: https://doi.org/10.1007/978-1-0716-0892-0_3
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