Abl kinase constructs expressed in bacteria

Facilitation of structural and functional studies including segmental labeling by expressed protein ligation

Rong Xu, Dongsheng Liu, David Cowburn

Research output: Contribution to journalArticle

4 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1878-1885
Number of pages8
JournalMolecular BioSystems
Volume8
Issue number7
DOIs
StatePublished - Jul 2012

Fingerprint

Ligation
Phosphotransferases
Bacteria
Proteins
Protein-Tyrosine Kinases
src Homology Domains
Baculoviridae
X Ray Crystallography
Insects
Pathology
Escherichia coli

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Biology

Cite this

@article{3e8c2e17b4d04ccaa9932bbe41a68c0c,
title = "Abl kinase constructs expressed in bacteria: Facilitation of structural and functional studies including segmental labeling by expressed protein ligation",
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.",
author = "Rong Xu and Dongsheng Liu and David Cowburn",
year = "2012",
month = "7",
doi = "10.1039/c2mb25051a",
language = "English (US)",
volume = "8",
pages = "1878--1885",
journal = "Molecular BioSystems",
issn = "1742-206X",
publisher = "Royal Society of Chemistry",
number = "7",

}

TY - JOUR

T1 - Abl kinase constructs expressed in bacteria

T2 - Facilitation of structural and functional studies including segmental labeling by expressed protein ligation

AU - Xu, Rong

AU - Liu, Dongsheng

AU - Cowburn, David

PY - 2012/7

Y1 - 2012/7

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84862158283&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84862158283&partnerID=8YFLogxK

U2 - 10.1039/c2mb25051a

DO - 10.1039/c2mb25051a

M3 - Article

VL - 8

SP - 1878

EP - 1885

JO - Molecular BioSystems

JF - Molecular BioSystems

SN - 1742-206X

IS - 7

ER -