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Published on: December 24, 2016
Abl kinase constructs expressed in bacteria: facilitation of structural and functional studies including segmental
Rong Xu1, Dongsheng Liu, David Cowburn
1Dept. of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461, USA. rong.xu@einstein.yu.edu
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.
Insights
Researchers developed a straightforward method for producing purified Abelson kinase (Abl) protein in bacteria. This advance facilitates structural studies and the development of new molecular therapeutics for oncology and developmental pathologies.
Area of Science:
- Biochemistry and Structural Biology
- Molecular Therapeutics
- Oncology
Background:
- Tyrosine kinases play crucial roles in cell development and are implicated in cancer and developmental diseases.
- Targeting tyrosine kinases with molecular therapeutics is a major focus in drug discovery.
- Economical production of purified tyrosine kinases is essential for biochemical and structural studies but challenging in bacterial systems.
Purpose of the Study:
- To present a straightforward method for expressing and purifying Abelson kinase (Abl) protein domains in a bacterial system.
- To demonstrate the biological activity and correct folding of the expressed Abl protein.
- To showcase the utility of this method for segmental isotopic labeling in NMR structure-activity relationship studies.
Main Methods:
- Expression and purification of isolated Abl kinase domain and SH3-SH2-kinase multi-domain structures in a bacterial system.
- Assessment of protein folding and biological function.
- Application of expressed protein ligation for segmental isotopic labeling in bacterial-expressed Abl kinase domain constructs.
Main Results:
- A straightforward method for expressing and purifying Abl kinase and multi-domain constructs was successfully developed.
- The expressed Abl protein demonstrated correct folding and retained biological function.
- Soluble protein yields reached several mg L(-1) in minimal media, and segmental isotopic labeling was achieved.
Conclusions:
- This bacterial expression and purification method provides an economical and efficient approach for obtaining functional Abl kinase domains.
- The method facilitates crucial biochemical and structural studies, including NMR-based structure-activity relationship investigations.
- This advance supports the development of novel molecular therapeutics targeting tyrosine kinases in oncology and developmental pathologies.
