Proregion of Bombyx mori cysteine proteinase functions as an intramolecular chaperone to promote proper folding of the mature enzyme.
A cDNA encoding the proform of Bombyx cysteine proteinase (BCP) was expressed at a high level in Escherichia coli using the T7 polymerase expression system. The insoluble recombinant zymogen was solubilized and renatured by modifying a method applied to human pro-cathepsin L. Like the natural BCP precursor, the recombinant proenzyme was spontaneously converted to an active proteinase at pH 3.75. A deletion in the central region of the propeptide resulted in much loss of the activity, suggesting that the propeptide is essential for proper folding during renaturation. In contrast, the renatured mature form of recombinant BCP was not active but regained activity by including the propeptide in the renaturing buffer, suggesting that the propeptide, acting as an intramolecular chaperone, promotes refolding of the associated proteinase domain into an active conformation. The mature form of natural BCP rapidly lost its activity at neutral pH, whereas its proform was stable. The mature enzyme retained some activity in the presence of the propeptide. Arch.
Yamamoto
Y
Y
Department of Biochemistry and Radiation Biology, College of Agriculture, Yamaguchi University, Yamaguchi, Japan. yamataka@po. yb.cc.yamaguchi-u.ac.jp
Watabe
S
S
Kageyama
T
T
Takahashi
S Y
SY
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D004792:Enzyme Precursors; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases