Simplified protein purification using protein-polysaccharide conjugation.

摘要

The purification step of protein production and isolation is the most time consuming and complex step in processing. Optimization of the purification process requires rapid, economical, and cost-effective methods to purify proteins at both the small-scale and the large-scale. Instead of using complicated chromatographic techniques and dialysis, a more simplified method is proposed utilizing only a centrifuge. A tyrosine tag with an enterokinase enzymatic cleavage site was added to the C-terminus of green fluorescent protein (GFP). The tyrosine tagged protein was expressed in E. coli cells. The cells were harvested and lysed. The lysate containing the tyrosine tagged GFP was covalently coupled to a polysaccharide (chitosan) using tyrosinase enzyme. The GFP in the protein-polysaccharide conjugate was then liberated using the enzyme, enterokinase. This method was effective in conjugating and purifying the protein using a centrifuge with a limited number of processing steps.