Overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system

Abstract

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.

Functional and Structural Relationship between the Calmodulin-binding, Actin-binding, and Actomyosin-ATPase Inhibitory Domains on the C Terminus of Smooth Muscle Caldesmon

“…Transfection and Isolation of Recombinant Baculovirus-Production of baculovirus recombinants by co-transfecting Sf9 cells with a mixture of recombinant baculovirus vector and wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA and isolation of the recombinants were carried out as described (31,32,36,37).…”

“…Protein Purification-Recombinant full-length CaD and CaD mutants were prepared as described (36). Smooth muscle actin, CaD, myosin, and tropomyosin were purified from chicken gizzard (38 -40).…”

“…However, the exact location of the functional motifs in the sequence between residues 690 -717 and its structural relationship with CaM-binding site B are unknown (36). To further define the functional motifs, we constructed several internal deletion mutants of chicken gizzard smooth muscle CaD, which progressively lacked the targeted sequences within residues 690 -717, and overexpressed them in a baculovirus system (36). Fig.…”

“…1 illustrates the schematic structures of these internal mutants. All the mutant proteins were purified to near homogeneity (Ͼ95% purity) using our published method (36).…”

“…Inhibition of actin-(A) or tropomyosin-actin-(B) activated myosin ATPase activity by CaD (q), CaD⌬690 -717 (E), CaD⌬710 -717 (å), CaD⌬700 -717 (Ç), and CaD⌬690 -699 (f) was measured as described (36). Smooth muscle myosin and actin were kept constant at 1 and 25 M, respectively, and tropomyosin was mixed with actin at a 1:4 molar ratio.…”