Production of crystallizable human chymase from a Bacillus subtilis system.
A Bacillus subtilis strain deficient in seven extracellular proteases was used to produce human mast cell chymase and is a viable expression system for serine proteases and other classes of proteins. Chymase is produced at 0.3-0.5 mg/l and is purified by three chromatography steps. Two crystal forms of PMSF-treated chymase were optimized. The first is C2 with a=47.94 A, b=85.23 A, c=174.18 A, beta=96.74 degrees, and diffracts to at least 2.1 A, while the second is P212121, with cell dimensions a=43.93 A, b=58.16 A, and c=86.09 A, and a diffraction limit of approximately 1.9 A. The first crystal form has either three or four molecules/asymmetric unit, while the second has one molecule/asymmetric unit.
McGrath
M E
ME
Arris Pharmaceutical, South San Francisco, CA 94080, USA. mcgrath@arris.com
Osawa
A E
AE
Barnes
M G
MG
Clark
J M
JM
Mortara
K D
KD
Schmidt
B F
BF
D000595:Amino Acid Sequence; D001412:Bacillus subtilis; D001483:Base Sequence; D002845:Chromatography; D053818:Chymases; D003460:Crystallization; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008407:Mast Cells / Q000201:enzymology; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D053818:Chymases