Heterologous expression, purification, and properties of a potato protein inhibitor of serine proteinases.
The gene PKPI-B10 [AF536175] encoding in potato (Solanum tuberosum L., cv. Istrinskii) a Kunitz-type protein inhibitor of proteinases (PKPI) has been cloned into the pET23a vector and then expressed in Escherichia coli. The recombinant protein PKPI-B10 obtained as inclusion bodies was denatured, separated from admixtures by ion-exchange fast protein liquid chromatography (FPLC) on MonoQ under denaturing conditions, and renatured. The native protein was additionally purified by ion-exchange FPLC on DEAE-Toyopearl. The PKPI-B10 protein effectively inhibits the activity of trypsin, significantly weaker suppresses the activity of chymotrypsin, and has no effect on other serine proteinases: human leukocyte elastase, subtilisin Carlsberg, and proteinase K, and also the plant cysteine proteinase papain.
Speranskaya
A S
AS
Bach Institute of Biochemistry, Russian Academy of Sciences, 119071 Moscow, Russia.
Krinitsina
A A
AA
Revina
T A
TA
Gerasimova
N G
NG
Keruchen'ko
Ya S
YS
Shevelev
A B
AB
Valueva
T A
TA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011198:Solanum tuberosum / Q000235:genetics*; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors