Rice cystatin: bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein.
A cDNA clone that encodes oryzacystatin, a cysteine protease inhibitor from rice, was isolated and expressed in Escherichia coli BL-21 (DE3) using an expression plasmid under the control of a T7 RNA polymerase promoter. The construct pT7OC 9b encoded a fusion protein containing 11 amino acid residues of the NH2 terminus of the bacterial protein phi 10 and 79 residues of oryzacystatin lacking 23 NH2-terminal residues of the wild-type protein. Recombinant oryzacystatin (ROC) constituted approximately 10% of the total bacterial protein mass and was purified in a single step by anion-exchange chromatography. The inhibitory activity of ROC toward papain (Ki = 3 x 10(-8) M) was comparable with that of the naturally occurring protein isolated from rice. Caseinolytic activity in midgut homogenates from seven species of stored product insects was inhibited from 18 to 85% by ROC, whereas the same activity was inhibited from 14 to 69% by the serine proteinase inhibitor phenylmethylsulfonyl fluoride. Midguts of stored product insects apparently contain both cysteine proteinases and serine proteinases, but the relative amounts vary with the species. When fed to the red flour beetle, Tribolium castaneum, 10 wt% ROC in the diet suppressed growth approximately 35% relative to that of the control group of insects.
Chen
M S
MS
Department of Biochemistry, Kansas State University, Manhattan 66506.
Johnson
B
B
Wen
L
L
Muthukrishnan
S
S
Kramer
K J
KJ
Morgan
T D
TD
Reeck
G R
GR
D000818:Animals; D001483:Base Sequence; D001517:Coleoptera / Q000187:drug effects / Q000254:growth & development; D015891:Cystatins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D004247:DNA / Q000235:genetics; D004926:Escherichia coli; D006131:Growth Inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology*; D010206:Papain / Q000037:antagonists & inhibitors; D010446:Peptide Fragments / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology
D015891:Cystatins; D006131:Growth Inhibitors; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D004247:DNA; D010206:Papain