Subcloning, expression and purification of Human Hialuronidase-1, variant 8
Del Monaco ADM, Hirata MH
Hyaluronic Acid, HA is a major component of the extracellular matrix of vertebrates. It is a glycosaminoglycan hydrolyzed by enzymes of the hyaluronidase family, involved in the regulation of important biological processes such as angiogenesis and vascular permeability. As interest in the development of a synthesis route for this enzyme, we aim to obtain a plasmid containing the coding sequence of gene variant 8 Hyal-1. To obtain the plasmid insert was planned and two restriction sites for sub-cloning site directed at the 5 'Bam H-1' and 3 'Not-1 in codon sequence of Hyal-1. The insert was sub-cloned into plasmid pET28-a, and transfected for expression in Escherichia coli Bl-21. The expression was induced by IPTG in best time of 4 hours and confirmation of protein expression was performed by Western blotting. There was a 45 kDa protein, thus confirming the presence of Hyal-1. Purification was performed on nickel agarose column to obtain a larger amount of the protein, approximately 25μg/L. The route suggested in this study was efficient attainment of Hyal-1 recombinant protein.
Palavras-chaves: Subcloning, Recombinant proteins, Hyaluronic acid, Hialuronidase enzyme