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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).

Original publication

DOI

10.1007/978-1-0716-0892-0_3

Type

Journal article

Journal

Methods in molecular biology (Clifton, N.J.)

Publication Date

01/2021

Volume

2199

Pages

23 - 43

Addresses

Diamond Light Source Ltd., Didcot, Oxfordshire, UK.