[16] Expression of protein kinase Cγ regulatory domain elements containing cysteine-rich zinc-coordinating regions as glutathione S-transferase fusion proteins

This chapter describes studies that were initially undertaken to optimize expression conditions for the cysteine-rich regions in XLIB E. coli and subsequent modifications thereof for the expression of the entire regulatory domain in BL21 E. coli. Protein kinase C (PKC) is a family of serine/threonine protein kinases consisting of 10 or more members, whose activities are physiologically regulated by lipids. Members of the PKC family are divided into three groups based on cDNA-derived protein sequence similarities: calcium-dependent (cPKCs), non-calcium-dependent (nPKCs), and atypical (aPKCs) PKC enzymes. Each isoform possesses two functionally distinct segments, a COOH-terminal kinase domain and an NH2-terminal regulatory domain. Each isoform possesses two functionally distinct segments, a COOH-terminal kinase domain and an NH2-terminal regulatory domain. The close proximity of several lipid interaction sites and the complexity of lipid interactions required for PKC activation have hampered the assessment of individual contributions toward PKC activation. The chapter discusses here how conditions that enable the expression and isolation of large quantities of segments of the PKCγ regulatory domain as soluble GST fusion proteins were initially established using XL1B as an E. coli host.