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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 SGC, we opted for the Ligation-Independent Cloning (LIC) method which provides the medium throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in a 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

10.1007/978-1-62703-691-7_4

Type

Journal article

Journal

Methods Mol Biol

Publication Date

2014

Volume

1091

Pages

55 - 72

Keywords

Cloning, Molecular, Escherichia coli, Genetic Vectors, Humans, Proteins, Recombinant Proteins, Transformation, Bacterial