Production and characterization of recombinant guamerin, an elastase-specific inhibitor, in the methylotrophic yeast Pichia pastoris.
The elastase-specific inhibitor, guamerin, was expressed and secreted into a culture medium using the methylotrophic yeast Pichia pastoris, and the resulting recombinant guamerin was purified from the culture media using a two-step procedure composed of a hydrophobic interaction and reverse-phase chromatography. Up to 90 g/L of dry cell weight, the guamerin-producing recombinant P. pastoris was cultivated and guamerin was secreted into the culture medium at a level of 0.69 g/L. The recombinant guamerin was highly purified (>98%) with a recovery yield of 68%. Analyses of the purified guamerin revealed the same N-terminal amino acid sequence, amino acid composition, and molecular mass as found in the native leech protein. The recombinant guamerin exhibited the tight binding to porcine pancreatic elastase. Furthermore, the recombinant guamerin did not produce a humoral immune response in mice.
Kim
K Y
KY
Mogam Biotechnology Research Institute, 341 Pojung-Ri, Koosung-Myun, Yongin, Kyonggi-Do, 449-910, Korea.
Lim
H K
HK
Lee
K J
KJ
Park
D H
DH
Kang
K W
KW
Chung
S I
SI
Jung
K H
KH
D000595:Amino Acid Sequence; D001483:Base Sequence; D002853:Chromatography, Liquid; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007447:Invertebrate Hormones / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013696:Temperature
D004791:Enzyme Inhibitors; D007447:Invertebrate Hormones; D011994:Recombinant Proteins; C093975:guamerin; D010196:Pancreatic Elastase