Recombinant Saccharomyces cerevisiae Phosphatidylinositol N-acetylglucosaminyltransferase GPI3 subunit (SPT14), partial

The production of the recombinant saccharomyces cerevisiae phosphatidylinositol N-acetylglucosaminyltransferase GPI3 subunit (SPT14) involves several steps in an E. coli expression system. Firstly, the gene fragment encoding the 1-377aa of the saccharomyces cerevisiae SPT14 protein alone with an N-terminal 10xHis-tag and a C-terminal Myc-tag is inserted into an appropriate expression vector. The resulting recombinant construct vector is then introduced into E. coli cells through a transformation process. Following transformation, the positive cells are selected, and a large-scale culture is established to facilitate protein expression. The N-terminal 10xHis-tag and C-terminal Myc-tag serve to facilitate the purification and detection of the recombinant SPT14 protein. After cell lysis, the protein is purified from cell lysate. The purity of the recombinant SPT14 protein is confirmed to be up to 90% using SDS-PAGE analysis. On the gel, the purified recombinant SPT14 protein is visualized as a distinct band with an estimated molecular weight of approximately 50 kDa.