Abstract
Human Tousled-like kinases (TLKs) are highly conserved serine/threonine protein kinases responsible for cell proliferation, DNA repair, and genome surveillance. Their possible involvement in cancer via efficient DNA repair mechanisms have made them clinically relevant molecular targets for anticancer therapy. Innovative approaches in chemical biology have played a key role in validating the importance of kinases as molecular targets. However, the detailed understanding of the protein structure and the mechanisms of protein–drug interaction through biochemical and biophysical techniques demands a method for the production of an active protein of exceptional stability and purity on a large scale. We have designed a bacterial expression system to express and purify biologically active, wild-type Human Tousled-like Kinase 1B (hTLK1B) by co-expression with the protein phosphatase from bacteriophage λ. We have obtained remarkably high amounts of the soluble and homogeneously dephosphorylated form of biologically active hTLK1B with our unique, custom-built vector design strategy. The recombinant hTLK1B can be used for the structural studies and may further facilitate the development of new TLK inhibitors for anti-cancer therapy using a structure-based drug design approach.
Highlights
Cancer is considered to be a severe health concern worldwide[1]
The most common method employed for the expression of the active forms of Human Tousled-like kinases is from the insect cell culture[11] or with minimal yields in bacteria[12]
For the first time, we have described in detail the soluble prokaryotic and homogeneously unphosphorylated expression of full-length, wild-type recombinant 6x His-tagged Human Tousled-like Kinase 1B (hTLK1B) using a bacteriophage λ phosphatase co-expression strategy and its simple purification by conventional immobilised metal affinity chromatography technique
Summary
Deregulation of the kinase activity has emerged as a primary mechanism by which cancer cells evade normal physiological constraints on the growth and survival. The Tousled-Like Kinases (TLK1 and TLK2) are highly conserved serine/threonine protein kinases involved in DNA repair, chromatin assembly, transcription and chromosome segregation[3]. Their discernible role in the genome surveillance, and in cancer development via efficient DNA repair mechanisms has been well substantiated[3]. The most common method utilised for the expression of the active forms of Human Tousled-like kinases (both TLK1 and TLK2) is from the insect cell culture[11] or with minimal yields in bacteria[12]. We report a method for bacterial expression and purification of the wild-type hTLK1B by co-expressing it along with bacteriophage lambda protein phosphatase
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