TITLE: Subcloning, expression and purification of Human Hialuronidase-1, variant 8
AUThOR(s): Del Monaco, A.D.M. & Hirata, M.H.
ABSTRACT: 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.
KEYWORDS: Subcloning, Recombinant proteins, Hyaluronic acid, Hialuronidase enzyme.
CITATION: Del Monaco A. D. M. & Hirata M. H. Subcloning, expression and purification of Human Hialuronidase-1, variant 8. The Academic Society Journal, 3(1) 50-63, 2019. DOI: doi.org/10.32640/tasj.2019.1.50.