OVEREXPRESSION, PURIFICATION, AND CHARACTERIZATION OF FULL-LENGTH ANDMUTANT CALDESMONS USING A BACULOVIRUS EXPRESSION SYSTEM

Three recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (PvlCaD), the full-l ength CaD codon sequence and a six-histidine tag at the 5'-end (pBlueB acHisCaD), or the full-length CaD codon sequence and an extra six-hist idine codon sequence at the 3'-end (PvlHisCaD) were constructed. Spodo ptera frugiperda (Sf9) cells transfected with these constructs overexp ressed full-length CaD, yielding 2, 20, and 50 mu g per 10(6) cells fo r pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course ass ays for the expressed proteins demonstrated that the optimum harvest t ime was 36 h postinfection. Immunofluorescence microscopy revealed Pvl CaD localized on the plasma membrane of Sf9 cells at 24 h postinfectio n and distributed throughout the cytoplasm at 36-48 h postinfection. A nalysis of the purified recombinant full-length CaD revealed most of t he characteristics of the authentic CaD, including (a) an electrophore tic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to i nhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of Ca2+-calmodulin to reverse the inhibition. A CaD mutant wi th a deletion of 159 amino acids from the carboxyl terminus of the ful l-length CaD was also expressed at high levels in Sf9 cells. However, this mutant showed a decreased ability to bind to actin, tropomyosin-a ctin, and calmodulin, whereas the myosin binding was unaffected; actin -activated ATP hydrolysis by smooth muscle myosin was not inhibited by this mutant.