Overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system.
Three recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (Pv1CaD), the full-length CaD codon sequence and a six-histidine tag at the 5'-end (pBlueBacHisCaD), or the full-length CaD codon sequence and an extra six-histidine codon sequence at the 3'-end (PvlHisCaD) were constructed. Spodoptera frugiperda (Sf9) cells transfected with these constructs overexpressed full-length CaD, yielding 2, 20, and 50 micrograms per 10(6) cells for pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course assays for the expressed proteins demonstrated that the optimum harvest time was 36 h postinfection. Immunofluorescence microscopy revealed PvlCaD localized on the plasma membrane of Sf9 cells at 24 h postinfection and distributed throughout the cytoplasm at 36-48 h postinfection. Analysis of the purified recombinant full-length CaD revealed most of the characteristics of the authentic CaD, including (a) an electrophoretic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to inhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of Ca(2+)-calmodulin to reverse the inhibition. A CaD mutant with a deletion of 159 amino acids from the carboxyl terminus of the full-length CaD was also expressed at high levels in Sf9 cells. However, this mutant showed a decreased ability to bind to actin, tropomyosin-actin, and calmodulin, whereas the myosin binding was unaffected; actin-activated ATP hydrolysis by smooth muscle myosin was not inhibited by this mutant.
Wang
Z
Z
Department of Pathobiology, University of Pennsylvania, Philadelphia 19104.
Horiuchi
K Y
KY
Jacob
S S
SS
Gopalakurup
S
S
Chacko
S
S
D000199:Actins / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002645:Chickens / Q000235:genetics; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000032:analysis; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009218:Myosins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D018411:Spodoptera; D014335:Tropomyosin / Q000378:metabolism
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D003062:Codon; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014335:Tropomyosin; D009218:Myosins