High yield bacterial expression, purification and characterisation of bioactive Human Tousled-like Kinase 1B involved in cancer

• Published in: Scientific reports 2018-03, Vol.8 (1), p.4796-9, Article 4796
• Main Authors: Bhoir, Siddhant, Shaik, Althaf, Thiruvenkatam, Vijay, Kirubakaran, Sivapriya
• Format: Article
• Language: English
• Subjects: 631/136/334/2241/1474; 631/1647/2196/2197; 631/1647/2230/2233; 631/1647/527/873; 631/326/1321; 631/337/1427/2566; 631/45/275; 82; 82/1; 82/16; 82/58; 82/80; 82/81; 82/83; 96; Adenosine Diphosphate - metabolism; Bacteriophage lambda - enzymology; Biological activity; Cancer; Cell proliferation; Deoxyribonucleic acid; DNA; DNA repair; Drug development; Drug interaction; Drug interactions; Escherichia coli - genetics; Escherichia coli - metabolism; Genomes; Humanities and Social Sciences; Humans; Kinases; Medical innovations; multidisciplinary; Phosphoprotein Phosphatases - genetics; Phosphoprotein Phosphatases - metabolism; Protein kinase; Protein phosphatase; Protein Serine-Threonine Kinases - genetics; Protein Serine-Threonine Kinases - isolation & purification; Protein Serine-Threonine Kinases - metabolism; Protein structure; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Science; Science (multidisciplinary); Serine; Threonine; Viral Proteins
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-018-22744-5

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.