title: Expression and purification of the N-terminal regulatory domain of Protein Kinase C for biophysical studies.
abstract: We report the protocol for heterologous expression and purification of the N-terminal regulatory region of two Protein Kinase C (PKC)(1) isozymes, one conventional and one novel. Previous studies of these domains relied almost exclusively on the fusion constructs with high-molecular-weight solubility fusion partners such as GST and MBP. We developed experimental procedures that enabled us to overcome challenges associated with the amphiphilic character of the regulatory domain and generate sufficient quantities of fusion partner-free proteins for biophysical work. The key features of the protocol are the identity of the cleavable fusion partner, expression conditions, growth medium additives, introduction of mutation/solubility tags, and incorporation of osmolytes. The protein yields are sufficient for cost-effective production of isotopically enriched proteins for NMR work and biophysical studies in general. Our work opens up an avenue for the structural studies of these challenging proteins with high amphiphilic character.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, 300 Olsen Boulevard, College Station, TX 77843, USA.
authors.forename: Taylor R
authors.initials: TR
authors.lastname: Cole
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, 300 Olsen Boulevard, College Station, TX 77843, USA. Electronic address: tigumenova@tamu.edu.
authors.forename: Tatyana I
authors.initials: TI
authors.lastname: Igumenova
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D009994:Osmolar Concentration; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011493:Protein Kinase C / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D051571:Protein Kinase C-alpha / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D051745:Protein Kinase C-delta / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment
chemical_list: D011993:Recombinant Fusion Proteins; C087857:protein kinase C gamma; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta
keywords: C1 domain; C2 domain; NMR spectroscopy; Osmolyte; Protein Kinase C