Expression and characterization of chicken ovoinhibitor in Pichia pastoris.
Chicken ovoinhibitor cDNA was prepared by reverse transcriptase-polymerase chain reaction (RT-PCR) using chicken oviduct mRNA. The ovoinhibitor cDNA was successfully cloned downstream from the AOXI promoter of pPICZalphaA plasmid vector to facilitate its expression in the methylotrophic yeast Pichia pastoris. The pPICZalphaA carrying the ovoinhibitor cDNA was integrated into the Pichia genome. The secreted recombinant ovoinhibitor was purified by ion-exchange chromatography on a DEAE sepharose column. The recombinant ovoinhibitor had a molecular mass of 49 kDa, as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and time of flight-mass spectrometry (TOF-MS) analyses. The recombinant ovoinhibitor, just as the native ovoinhibitor, showed inhibitory activity against trypsin, chymotrypsin and elastase.
Begum
Shamima
S
Department of Biological Chemistry, Yamaguchi University, Yamaguchi 753-8515, Japan.
Saito
Akira
A
Kato
Akio
A
He
Jianwei
J
Azakami
Hiroyuki
H
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry*; D015387:Egg Proteins, Dietary / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D018076:DNA, Complementary; D015387:Egg Proteins, Dietary; D011994:Recombinant Proteins; C004224:ovoinhibitor