Use of Pichia pastoris for the expression, purification, and characterization of rat calretinin "EF-hand" domains

Calretinin (CR) is a calcium-binding, neuronal protein of undefined functio n. Related proteins either buffer intracellular calcium concentrations or a re involved in calcium-signaling pathways. We transformed three CR gene fra gment sequences, corresponding to its three complementary domains (I-II, II I-IV, and V-VI), into Pichia pastoris. High yields of extracellular express ion, of more than 200 mg/liter, were achieved. Simple purification protocol s provide high yields of homogenous proteins: dialysis and DE-AE-cellulose chromatography for domains I-II and III-IV or ammonium sulfate precipitatio n and octyl-Sepharose chromatography for domain V-VI. To our knowledge, thi s is the first report of the expression of an EF-hand protein using P. past oris. Direct comparison of the purified yields of domain I-II indicates a s imilar to 20-fold improvement over Escherichia coli. N-terminal amino acid sequencing confirmed our gene products and two anti-calretinin antibodies r ecognized the appropriate domains. All three CR domains bind Ca-45 and the domain containing EF-hands V and VI seems to have a lower calcium capacity than the other domains. Circular dichroism indicates a high helix content f or each of the domains. Calcium-induced structural changes in the first two domains, followed by tryptophan fluorescence, correspond with previous stu dies, while tyrosine emission fluorescence indicates calcium-induced struct ural changes also occur in domain V-VI. The methods and expression levels a chieved are suitable for future NMR labeling of the proteins, with N-15 and C-13, and structure-function studies that will help to further understand CR function. (C) 1999 Academic Press.