Purification and Characterization of the Human Cysteine-Rich S100A3 Protein and Its Pseudo Citrullinated Forms Expressed in Insect Cells
High quantity and quality of recombinant Ca2+-binding proteins are required to study their molecular interactions, self-assembly, posttranslational modifications, and biological activities to elucidate Ca2+-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 Ca2+-affinity. Three free thiol groups located at the C-terminus are predicted to form the high-affinity Zn2+-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.
Key wordsBaculovirus Disulfide bridge Insect cell Peptidylarginine deiminase S100A3 Calcium-binding Zinc-binding EF-hand
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