Medium-Throughput Production of Recombinant Human Proteins: Ligation-Independent Cloning
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).
Key wordsPCR Gene Ligation-independent cloning (LIC) Construct Protein Crystallography
We would like to thank all the SGC scientists (past and present) who contributed towards the development of the method. The SGC is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, Genome Canada, GlaxoSmithKline, Lilly Canada, the Novartis Research Foundation, Pfizer, Takeda, AbbVie, the Canada Foundation for Innovation, the Ontario Ministry of Economic Development and Innovation, and the Wellcome Trust.
- 4.Invitrogen (2010) Gateway® Technology: a universal technology to clone DNA sequences for functional analysis and expression in multiple systems. Invitrogen Life Technologies, CarlsbadGoogle Scholar
- 7.Clontech (2012) In-Fusion® HD cloning kit user manual. Clontech, Mountain ViewGoogle Scholar