PURIFICATION AND CHARACTERIZATION OF RECOMBINANT TOBACCO ETCH VIRUS PROTEASE

Abstract

The development of expression systems which permit the overproduction of large amounts of protein is a very important advancement in modern biotechnology. Recent developments in cloning have eased some of the traditional problems of obtaining high yields of pure product. New techniques enable the researcher to express and purify proteins of interest by fusing the expressed protein to a variety of commercially available affinity tags such as, f3-galactosidase, glutathione-S-transferase (GST) (Smith, D. 1988), and polyhistidine peptides (Guan, C. 1987). A new site specific protease, Tobacco Etch Virus protease (rTEV), has been cloned and overexpressed for the use in rapid cloning and purification procedures. In a purification scheme, the protein of interest will contain a TEV cleavage site flanked with six histidines. When the protein is cleaved only a single glycine residue at the amino terminus of the protein remains from the inserted rTEV site. With only one glycine residue left on the purified protein there is less of a chance for incorrect foldings, or for unexpected activities.