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Screening and production of recombinant human proteins: ligation-independent cloning

DOI: 10.1007/978-1-0716-0892-0_3 DOI Help

Authors: Claire Strain-damerell (Diamond Light Source) , Pravin Mahajan (Astex Pharmaceuticals) , Alejandra Fernandez-cid (Structural Genomics Consortium, University of Oxford) , Opher Gileadi (Structural Genomics Consortium, University of Oxford) , Nicola A. Burgess-brown (Structural Genomics Consortium, University of Oxford)
Co-authored by industrial partner: Yes

Type: Book Chapter

Book Chapter: 3

State: Published (Approved)
Published: October 2020

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).

Journal Keywords: PCR; Gene; Ligation-independent cloning (LIC); Construct; Protein; Crystallography

Subject Areas: Biology and Bio-materials

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Added On: 03/11/2020 09:46

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