title: Medium-throughput production of recombinant human proteins: ligation-independent cloning.
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 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).
authors.affiliation: Structural Genomics Consortium, University of Oxford, Oxford, UK.
authors.forename: Claire
authors.initials: C
authors.lastname: Strain-Damerell
authors.forename: Pravin
authors.initials: P
authors.lastname: Mahajan
authors.forename: Opher
authors.initials: O
authors.lastname: Gileadi
authors.forename: Nicola A
authors.initials: NA
authors.lastname: Burgess-Brown
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D014169:Transformation, Bacterial
chemical_list: D011506:Proteins; D011994:Recombinant Proteins