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Abl kinase constructs expressed in bacteria: facilitation of structural and functional studies including segmental labeling by expressed protein ligation

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Molecular BioSystems Vol - 8 Issue - 6

Molecular BioSystems Vol - 8 Issue - 5

Molecular BioSystems Vol - 8 Issue - 4

Molecular BioSystems Vol - 8 Issue - 3

Molecular BioSystems Vol - 8 Issue - 2

Molecular BioSystems Vol -7

Molecular BioSystems Vol -6

Molecular BioSystems Vol -5

Molecular BioSystems Vol -4

Molecular BioSystems Vol -3

Molecular BioSystems Vol -2

Abl kinase constructs expressed in bacteria: facilitation of structural and functional studies including segmental labeling by expressed protein ligation

Content Provider	Royal Society of Chemistry (RSC)
Author	Liu, Dongsheng Cowburn, David Xu, Rong
Copyright Year	2012
Abstract	A great portion of tyrosine kinases are involved in cell development and their structural alteration is intimately involved in associated pathologies of development and oncology. These kinases are one of the 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 levels 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−1 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.
Starting Page	1878
Ending Page	1885
Page Count	8
File Format	HTM / HTML PDF
ISSN	1742206X
Volume Number	8
Issue Number	7
Journal	Molecular BioSystems
DOI	10.1039/c2mb25051a
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	Tyrosine SH3 domain SH2 domain ABL (gene) Escherichia coli Oncology Abl Intein Protein X-ray crystallography Baculoviridae Kinase Nuclear magnetic resonance spectroscopy of proteins Abelson Insect Isotopic labeling
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Biotechnology

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

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Abl kinase constructs expressed in bacteria: facilitation of structural and functional studies including segmental labeling by expressed protein ligation

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