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 article
Methods in molecular biology (Clifton, N.J.)
01/2021
2199
23 - 43
Diamond Light Source Ltd., Didcot, Oxfordshire, UK.
Humans, Escherichia coli, Baculoviridae, Recombinant Proteins, Cloning, Molecular, Gene Expression, Genetic Vectors