title: Use of Pichia pastoris for the expression, purification, and characterization of rat calretinin "EF-hand" domains.
abstract: Calretinin (CR) is a calcium-binding, neuronal protein of undefined function. Related proteins either buffer intracellular calcium concentrations or are involved in calcium-signaling pathways. We transformed three CR gene fragment sequences, corresponding to its three complementary domains (I-II, III-IV, and V-VI), into Pichia pastoris. High yields of extracellular expression, of more than 200 mg/liter, were achieved. Simple purification protocols provide high yields of homogenous proteins: dialysis and DEAE-cellulose chromatography for domains I-II and III-IV or ammonium sulfate precipitation and octyl-Sepharose chromatography for domain V-VI. To our knowledge, this is the first report of the expression of an EF-hand protein using P. pastoris. Direct comparison of the purified yields of domain I-II indicates a approximately 20-fold improvement over Escherichia coli. N-terminal amino acid sequencing confirmed our gene products and two anti-calretinin antibodies recognized the appropriate domains. All three CR domains bind (45)Ca 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 for each of the domains. Calcium-induced structural changes in the first two domains, followed by tryptophan fluorescence, correspond with previous studies, while tyrosine emission fluorescence indicates calcium-induced structural changes also occur in domain V-VI. The methods and expression levels achieved are suitable for future NMR labeling of the proteins, with (15)N and (13)C, and structure-function studies that will help to further understand CR function.
authors.affiliation: Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, 3 Pasteur Street, Warsaw, 02-093, Poland.
authors.forename: M
authors.initials: M
authors.lastname: Palczewska
authors.forename: P
authors.initials: P
authors.lastname: Groves
authors.forename: J
authors.initials: J
authors.lastname: Kuznicki
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D064032:Calbindin 2; D002118:Calcium / Q000378:metabolism; D002845:Chromatography; D002942:Circular Dichroism; D020832:EF Hand Motifs; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010843:Pichia / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D064030:S100 Calcium Binding Protein G / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D020539:Sequence Analysis, Protein
chemical_list: C576512:Calb2 protein, rat; D064032:Calbindin 2; D010446:Peptide Fragments; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D002118:Calcium