Gene Cloning And Efficient Soluble Expression Of Human Adiponectin In Escherchia Coli

Adiponectin, as a specific adipokine, entails diverse biological roles associated with obesity, insulin resistance, type II diabetes and cardiovascular diseases, with broad potentials of application. At present, many researchers abroad commit themselves to studying the properties and functions of adiponectin, but the relevant work inland are rare.In this study, by using the total human blood genomic DNA as template, we particularly exploited the approach of splicing by overlap extension (SOE) to combine the two individually amplified exon fragments containing adiponectin-coding regions, and obtained the accurate, full-length of adiponectin gene (AP) verified by sequencing analysis. Subsequently, an expression construct pET-wAP was achieved by fusing the adiponectin gene behind the thioredoxin region within the potent plasmid pET-32a(+). The fusion form of thioredoxin-adiponectin was successfully expressed by IPTG induction, with a high yield up to 41% of the total cell protein but mainly in inclusion bodies.In order to achieve efficient and soluble expression of human adiponectin, we have optimized the expression constructs by using several consecutive strategies as follows. First, we fused human adiponectin gene (AP) behind the EC fragment with His₆ tag to get the recombinant plasmid pECAP. The fusion form of ECAP was successfully expressed by IPTG induction, with a yield up to 32.5% of the total cell protein, and the percentage of soluble fusion protein was 17.5%. Second, to further increase the solubility of the target protein, we used NusA as the fusion partner on the basis of pECAP to create the recombinant plasmid pNuECAP. The fusion form of NuECAP was successfully expressed by IPTG induction, with a high yield up to 39.5% of the total cell protein, and the percentage of soluble fusion protein was 30.7%. However, due to the large molecular weight of NusA, the net percentage of trimmed intact human adiponectin protein was not very considerable. Thereby, we finally introduced a synthetic rubredoxin gene as a fusion tag on the basis of pECAP...