Comparative of Recombinant Vespa affinis Hyaluronidase Expressed in Different Cloning Vector and their Biological Properties

Abstract

Cloning and expression of recombinant Vespa affinis hyaluronidase (rVesA2) were successfully expressed in Escherichia coli system. The VesA2 gene was cloned into pET-17b and pET-32a cloning vectors which had molecular weight 41.71 and 59.0 kDa, respectively. The recombinant plasmid of pET-17b was composed 1.08 kDa his-tag at the N-terminal. The 17.14 kDa of fusion tag; thioredoxin tag, histidine tag, and S-tag, was found in pET-32a. The verified expression conditions of rVesA2 induced under the conditions of 0.1 mM IPTG at 37â—¦C for 4 hrs gave the highest quantity of protein expression. The colony PCR and sequencing analysis were used to verify the rVesA2. The positive clones were detected the hyaluronidase activity by a zymographic gel. Recombinant proteins from both cloning vectors were insoluble. However, the recombinant from pET-32a showed higher solubility than that form pET-17b, after dissolving in 4 M urea solution. This result suggests that the fusion tag increase protein solubility.