Abl Kinase Constructs Expressed in Bacteria: facilitation of structural and functional studies including segmental labeling by expressed protein ligation

摘要

A great portion of tyrosine kinases are involved in cell development and their structural alteration are intimately involved in associated pathologies of development and oncology. These kinases are one of major groups of targets under investigation for molecular therapeutics. To carry out biochemical and structural biological studies on these kinases, economical production of their purified forms is highly desirable. However over-expressing tyrosine kinases as recombinant forms in bacterial systems and their purification is a significant challenge. Abelson kinase (Abl) has previously been expressed on a large scale to facilitate X-ray crystallography and NMR structure studies mainly in baculovirus infected insect cells. Even though success has been achieved in expression of soluble tyrosine kinases in E. coli with chaperones to improve correct folding, low expression level of kinases are intrinsic in such systems because of diversion of resources to produce chaperones. Here we present a straightforward method to express and purify isolated Abl kinase domain, and SH3-SH2-kinase multi-domain structures. The expressed Abl protein retains its correct folding and biological function. The yield of soluble protein is in a several mg/L range in minimal media. Furthermore we demonstrate that segmental isotopic labelling using expressed protein ligation can be achieved using bacterial expressed Abl kinase domain constructs, which is especially useful in NMR structure/activity studies.