Methods for the recombinant expression of active tyrosine kinase domains: Guidelines and pitfalls
AuthorsDíaz Galicia, M. Escarlet
Arold, Stefan T.
KAUST DepartmentBiological and Environmental Sciences and Engineering (BESE) Division
Computational Bioscience Research Center (CBRC)
Computer, Electrical and Mathematical Sciences and Engineering (CEMSE) Division
Online Publication Date2019-03-26
Print Publication Date2019
Permanent link to this recordhttp://hdl.handle.net/10754/631891
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AbstractProtein tyrosine kinases (PTKs) are key signaling molecules and important drug targets. Although the efficient recombinant production of active PTKs is important for both pharmaceutical industry and academic research, most PTKs are still obtained from conventional, expensive and time-consuming insect-cell based expression. Host toxicity, kinase inactivity, insolubility and heterogeneity are among the reasons thought to preclude PTK expression in Escherichia coli. Herein we review these presumed roadblocks and their possible solutions for bacterial expression of PTKs, and give an overview on kinase activity assays. Finally, we report our experiences and observations with the kinases Src, Lyn and FAK as examples to illustrate implementation, effects and pitfalls of E. coli expression and in vitro assaying of PTKs.
CitationDíaz Galicia ME, Aldehaiman A, Hong S, Arold ST, Grünberg R (2019) Methods for the recombinant expression of active tyrosine kinase domains: Guidelines and pitfalls. Methods in Enzymology. Available: http://dx.doi.org/10.1016/bs.mie.2019.02.027.
SponsorsThis research was supported by the King Abdullah University of Science and Technology (KAUST) through the baseline fund and the Award No. URF/1/2602-01 and FCC/1/1976-21 from the Office of Sponsored Research (OSR). Supplemental data, detailed methods and complete sequences are available on http://github.com/strubelab/kinaseexpress.
JournalMethods in Enzymology