Recombination And Expression Of SM22α And Its Deleted Mutant And Study Of Their Functions

Phenotypic modulation of vascular smooth muscle cell (VSMC) plays a key role in vascular diseases, such as atherosclerosis, hypertension and restenosis. Recent studies have focused on the expression regulation of VSMC-specific marker genes and cytoskeleton organization in association with phenotypic modulation of VSMC. Smooth muscle 22 alpha (SM22α) is a differentiated VSMC marker, which is characterized by its smooth muscle tissue-specific and VSMC phenotype-specific expression pattern. In addition, This protein is supposed to be associated with actin polymerization. To identify the physical interaction between SM22αand actin, their binding domains, and their roles in VSMC cytoskeleton reorganization and phenotypic modulation, prokaryotic expression system was established to express GST-SM22αfusion protein and GST-SM22α(1-151) fusion protein which is deleted ABS1 and CLR domains in E. coli. The purified proteins were used to in vitro observe their relationship with SMα-actin polymerization and from different phenotypic VSMC.1 Construction and identification of pGEX4T-SM22αand pGEX4T-SM22α(1-151)Prokaryotic expression plasmid of GST-SM22αand GST-SM22α(1-151) were constructed. The recombinant plasmids were identified by PCR amplification and EcoR I/Xho I digestion, and they were named as pGEX4T-SM22αand pGEX4T-SM22α(1-151), respectively.2 Expression of GST-SM22αand GST-SM22α(1-151) fusion proteinsGST-SM22αand GST-SM22α(1-151) fusion proteins could be expressed in pGEX4T-SM22αand pGEX4T-SM22α(1-151) hosted E. coli after induction by 0.5 mM IPTG for 6 h at 30℃, which served as soluble protein or inclusion bodies.3 Purification of GST-SM22αand GST-SM22α(1-151) fusion proteinsThe fusion proteins were purified from the supernatants of bacteria lysates via GST-Sepharose 4B affinity resin, 20 mg purified soluble GST-SM22αfusion protein was obtained from 100 ml bacteria culture.4 [KCl] influences SM22α-actin bindingTo evaluate whether KCl affects the binding of SM22αand actin in vitro, we perform affinity GST pull down assay and F-actin polymerization analysis in vitro using GST-SM22αor GST-SM22α(1-151) and actin in the different KCl concentration (25, 50, 100 and 150 mM). The results of GST pull down assays showed that SMα-actin binding to GST-SM22αin the affinity resin decreased with the increasing of [KCl]. F-actin polymerization analysis in vitro demonstrated that the crosslinking F-actin declined with the increasing of [KCl]. In summery, it is identified that [KCl] influences SM22α-actin interaction, which is decreased with the increasing [KCl].5 SM22αinteracts with actin via ABS1 and CLR domainsThe affinity GST pull-down assay was performed to investigate physical interaction between GST-SM22αor GST-SM22α(1-151) and actin. Briefly, F-actin fraction extracts from VSMC were incubated with glutathione Sepharose 4B beads coupled a recombinant GST-SM22αor GST-SM22α(1-151) fusion protein. The beads were washed, and interacting proteins were detected by Western blotting. SMα-actin was found in the affinity resin with GST-SM22α, but not GST-SM22α(1-151), suggesting physical interaction between SM22αand F-actin is by ABS1 and CLR domains. To further determine the activity of SM22αABS1 and CLR domains in vitro, purified GST-SM22αfusion protein or GST-SM22α(1-151) fusion protein and F-actin were incubated together, respectively. Protein in the pellets and supernatants were analyzed by SDS-PAGE. The crosslinking F-actin was found in the pellets with GST-SM22α, but not in the pellets with GST-SM22α(1-151). The results suggested that SM22αpromotes polymerization and cross-linking of F-actin in vitro via ABS1 and CLR domains. In summary, these results suggested that SM22αthough interacting with F-actin participates in polymerization and crosslinking of F-actin via ABS1 and CLR domains during VSMC phenotypic remodeling. Conclusions:1 E.coli expression system of GST-SM22αand GST-SM22α(1-151) fusion proteins are successfully constructed. Purified GST-SM22αfusion protein and GST-SM22α(1-151) fusion protein are isolated. 20 mg purified soluble fusion protein was obtained from 100 ml bacteria culture.2 KCl concentration influences SM22α-actin interacting, SM22α-actin interaction is decreased with the increasing of [KCl].3 SM22αABS1 and CLR domains physically interacts with actin and promotes polymerization and cross-linking of actin.