원문정보
초록
영어
Pancreatic or tissue carboxypeptidase B (CPB; EC 3.4.17.2) is a key enzyme involved in insulin conversion and highly specific for excising C-terminal Lys and Arg resiudes from peptides and proteins. Also, CPB is naturally synthesized in form of zymogen with an 11 kDa N-terminal prodomain that covers the catalytic pocket of the enzyme.1) A cDNA containing the human pancreatic pro-CPB was fused to three mutants of exoinulinase secretion signal (m1, m2,
and m3) from Kluyveromyces marxianus. We used these plasmids in order to further improve the secretion efficiency of proCPB. The recombinant plasmids, pYInum1-hproCPB, pYInum2-hproCPB, and pYInum3-hproCPB, were transformed into Saccharomyces cerevisiae 2805 strain. Yeast transformants were selected on the synthetic defined media lacking uracil. The recombinant hproCPBs were successfully expressed in S. cerevisiae after induction of
galactose, and could be secreted into the supernatant of the culture. The secretion efficiency of proCPBs from each vectors were measured and analyzed. Each the human proCPB protein secreted by three mutants of exoinulinase signal sequence was purified and the enzymatic properties were examined.