Purification of a cysteine protease inhibitor from larval hemolymph of the tobacco hornworm (Manduca sexta) and functional expression of the recombinant protein.
A cysteine protease inhibitor (CPI) with an apparent molecular mass of 11.5kDa was purified from larval hemolymph of the tobacco hornworm (Manduca sexta) by gel filtration on Sephadex G-50 followed by hydrophobic and ion-exchange column chromatographies. The purified cysteine proteinase inhibitor, denoted as MsCPI, strongly inhibited the plant cysteine protease, papain, with a K(i) value of 5.5 x 10(-9)M. Nucleotide sequence analysis of a partial cDNA encoding MsCPI indicated that MsCPI consists of 105 amino acid residues in a sequence that is similar to sarcocystatin A from Sarcophaga peregrina. However, northern blotting and PCR analyses using the specific primers of MsCPI suggested that the mRNA encoding MsCPI had a size of more than 12 kilobases, which included at least six tandemly repeated MsCPI segments. MsCPI was expressed in Escherichia coli and the recombinant protein effectively inhibited cysteine proteases from plants as well as from animals such as cathepsins B (K(i), 6.8 nM), H (3.0 nM), and L (0.87 nM). There was no inhibition exhibited toward trypsin, chymotrypsin, subtilisin, pepsin or themolysin.
Miyaji
Takayuki
T
Laboratory of Food Molecular Functionality, College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami-machi, Inashiki-gun, Ibaraki 300-0393, Japan.
Kouzuma
Yoshiaki
Y
Yaguchi
Jun
J
Matsumoto
Rika
R
Kanost
Michael R
MR
Kramer
Karl J
KJ
Yonekura
Masami
M
D000595:Amino Acid Sequence; D000818:Animals; D002403:Cathepsins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006458:Hemolymph / Q000737:chemistry*; D006801:Humans; D007814:Larva; D018402:Manduca / Q000254:growth & development*; D008969:Molecular Sequence Data; D010206:Papain / Q000037:antagonists & inhibitors; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; D011994:Recombinant Proteins; D002403:Cathepsins; D010206:Papain