Purification and characterization of recombinant rat mast cell protease 7 expressed in Pichia pastoris.
Rat mast cell protease 7 (rMCP7) is a neutral serine protease and a component of mast cells, where it is stored in secretory granules. Mast cells express numerous proteases so in order to characterize rMCP7, it was cloned and expressed as a recombinant protein in Pichia pastoris. During expression, rMCP7 protein was cleaved from the alpha-mating factor signal at the engineered KEX2 cleavage site to produce active rMCP7. The protein produced was stable at pH 5.5 and active in the absence of heparin. The rMCP7 was glycosylated and treatment with N-glycosidase F resulted in a protein of the predicted molecular mass of 30 kDa. The rMCP7 was purified via an ammonium sulfate precipitation, using casein as a carrier protein, followed by cation exchange chromatography. The purified protein was assayed using a range of substrates and where possible, k(m) and k(cat) values were determined. The substrate profile displayed by the recombinant rMCP7 was consistent with that of tryptase isolated from rat skin. The expression and purification of recombinant rMCP7 offer an efficient, low-cost method of producing large amounts of protein. It also offers the opportunity of easy manipulation and mutagenesis of rMCP7 for further biochemical, structural, and physiological studies.
Department of Molecular Biology, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire LE11 5RH, UK.
Carolyn
C
Lawson
Craig
C
Walker
James
J
Awford
Mark
M
Biffen
Philip
P
Mallinder
Andrew
A
Jackson
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D053818:Chymases; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D007700:Kinetics; D008407:Mast Cells / Q000201:enzymology*; D010843:Pichia / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D053818:Chymases