title: Purification and characterization of the human cysteine-rich S100A3 protein and its pseudo citrullinated forms expressed in insect cells.
abstract: High quantity and quality of recombinant Ca(2+)-binding proteins are required to study their molecular interactions, self-assembly, posttranslational modifications, and biological activities to elucidate Ca(2+)-dependent cellular signaling pathways. S100A3 is a unique member of the S100 protein family with the highest cysteine content (10%). This protein, derived from human hair follicles and cuticles, is characterized by an N-terminal acetyl group and irreversible posttranslational citrullination by peptidylarginine deiminase causing its homotetramer assembly. Insect cells, capable of introducing eukaryotic N-terminus and disulfide bonds, are an appropriate host in which to express this cysteine-rich protein. Four out of ten cysteines in the recombinant S100A3 form two intramolecular disulfide bridges that modulate its Ca(2+)-affinity. Three free thiol groups located at the C-terminus are predicted to form the high-affinity Zn(2+)-binding site. Citrullination of specific arginine residues in native S100A3 can be mimicked by site-directed mutagenic substitution of Arg/Ala. This chapter details our procedures used for the purification and characterization of the human S100A3 protein and its pseudo citrullinated forms expressed in insect cells.
authors.affiliation: Innovative Beauty Science Laboratory, Kanebo Cosmetics Inc., Odawara, Kanagawa, Japan. kizawa.kenji@kanebocos.co.jp
authors.forename: Kenji
authors.initials: K
authors.lastname: Kizawa
authors.forename: Masaki
authors.initials: M
authors.lastname: Unno
authors.forename: Hidenari
authors.initials: H
authors.lastname: Takahara
authors.forename: Claus W
authors.initials: CW
authors.lastname: Heizmann
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002956:Citrulline / Q000378:metabolism*; D003001:Cloning, Molecular; D003545:Cysteine; D018076:DNA, Complementary / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D059765:Homologous Recombination; D006801:Humans; D016296:Mutagenesis; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009418:S100 Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D018076:DNA, Complementary; D004274:DNA, Recombinant; D004220:Disulfides; D011994:Recombinant Proteins; D009418:S100 Proteins; C082356:S100A3 protein, human; D002956:Citrulline; D003545:Cysteine