All four repeating domains of the endogenous inhibitor for calcium-dependent protease independently retain inhibitory activity. Expression of the cDNA fragments in Escherichia coli.
We have already determined the primary structure of the endogenous inhibitor for calcium-dependent protease (CANP inhibitor, calpastatin) from the cDNA sequence and revealed that the CANP inhibitor contains four internally repeating units which could be responsible for its multiple reactive sites (Emori, Y., Kawasaki, H., Imajoh, S., Imahori, K., and Suzuki, K. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3590-3594). Restriction fragments of the cDNA corresponding to each of the four domains (encoding 104-156 amino acid residues of the total 718 residues) were subcloned into the multicloning site of pUC9 or pUC18 in a direction and frame matched to the lacZ' open reading frame of the vector. Under the lac operator-promoter system, we succeeded in producing truncated fragments of the CANP inhibitor in Escherichia coli. The CANP inhibitor fragments were partially purified, and the inhibitory activities toward calcium-dependent protease (CANP) were examined. All fragments containing well conserved regions of about 30 amino acid residues (domains I-IV) located in the middle of the four units exhibited the inhibitory activity. However, their inhibitory activities varied considerably. Further truncation experiments revealed that small fragments containing 30-70 amino acid residues of the CANP inhibitor still retained inhibitory activity. From these experimental results the following conclusions can be drawn: 1) each of the four repeating units of the CANP inhibitor (about 140 amino acid residues) is a real functional unit and can inhibit CANP activity independently; and 2) domains corresponding to well conserved sequences of about 30 amino acid residues containing a consensus Thr-Ile-Pro-Pro-X-Tyr-Arg sequence are essential for the inhibitory activity, and the bordering regions are important for its modulation.
Emori
Y
Y
Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Japan.
Kawasaki
H
H
Imajoh
S
S
Minami
Y
Y
Suzuki
K
K
D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002154:Calpain / Q000037:antagonists & inhibitors*; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid
D002135:Calcium-Binding Proteins; C033668:calpastatin; D004247:DNA; D002154:Calpain