Recombinant production of enzymatically active male contraceptive drug target hTSSK2 - Localization of the TSKS domain phosphorylated by TSSK2

• Published in: Protein expression and purification Vol. 121; pp. 88 - 96
• Main Authors: Shetty, Jagathpala, Sinville, Rondedrick, Shumilin, Igor A., Minor, Wladek, Zhang, Jianhai, Hawkinson, Jon E., Georg, Gunda I., Flickinger, Charles J., Herr, John C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2016
• Subjects: Baculoviridae - genetics; Caseins - metabolism; Contraceptive Agents, Male - pharmacology; Cytoskeletal Proteins; Enzyme Inhibitors - pharmacology; Gene Expression - drug effects; Humans; Male; Male contraceptive drug targets; Mobility shift assay; Phosphoproteins; Phosphorylation - drug effects; Protein Domains; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein Serine-Threonine Kinases - antagonists & inhibitors; Protein Serine-Threonine Kinases - biosynthesis; Protein Serine-Threonine Kinases - genetics; Protein Serine-Threonine Kinases - metabolism; Recombinant kinases; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Staurosporine - pharmacology; TSKS; TSSK2; TSKS; TSSK; SDS-PAGE; Male contraceptive drug targets; IMAC; Mobility shift assay; Recombinant kinases; E.coli; MSA; EDTA; TSSK2; Ni-NTA
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2016.01.009

The testis-specific serine/threonine kinase 2 (TSSK2) has been proposed as a candidate male contraceptive target. Development of a selective inhibitor for this kinase first necessitates the production of highly purified, soluble human TSSK2 and its substrate, TSKS, with high yields and retention of biological activity for crystallography and compound screening. Strategies to produce full-length, soluble, biologically active hTSSK2 in baculovirus expression systems were tested and refined. Soluble preparations of TSSK2 were purified by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography. The biological activities of rec.hTSSK2 were verified by in vitro kinase and mobility shift assays using bacterially produced hTSKS (isoform 2), casein, glycogen synthase peptide (GS peptide) and various TSKS peptides as target substrates. Purified recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay by phosphorylating hTSKS isoform 2 and casein. The ATP Km values were similar for highly and partially purified fractions of hTSSK2 (2.2 and 2.7 μM, respectively). The broad spectrum kinase inhibitor staurosporine was a potent inhibitor of rec.hTSSK2 (IC50 = 20 nM). In vitro phosphorylation experiments carried out with TSKS (isoform 1) fragments revealed particularly strong phosphorylation of a recombinant N-terminal region representing aa 1–150 of TSKS, indicating that the N-terminus of human TSKS is phosphorylated by human TSSK2. Production of full-length enzymatically active recombinant TSSK2 kinase represents the achievement of a key benchmark for future discovery of TSSK inhibitors as male contraceptive agents. •Soluble full-length hTSSK2 was produced in a baculovirus expression system and purified.•The biological activity of the recombinant hTSSK2 was studied by in vitro kinase and mobility shift assays.•Recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay.•The ATP Km values were similar for the highly and partially purified fractions of hTSSK2.•In vitro phosphorylation experiments revealed that the N-terminus of hTSKS is phosphorylated by hTSSK2.