278013 Protein Purification Using Lytag and Q Sepharose
Protein Purification using LYTAG and Q Sepharose
A method of purifying recombinant proteins using affinity chromatography with Q Sepharose and a LYTAG-intein affinity tag was developed. The LYTAG protein affinity tag is currently used with a specifically designed resin for recombinant protein purification. Inteins are proteins that have been engineered to cleave on a shift in pH or temperature, facilitating their use as a component of self-cleaving tags. The gene that creates LYTAG is joined with the gene for a self-cleaving intein, and this combined gene is joined to the gene for an interchangeable product protein. The cell then reads the genes and creates proteins, called fusion proteins, made of LYTAG connected to intein and an interchangeable product protein to be purified. The LYTAG-intein fusion proteins can be attached to the Q Sepharose resin. The intein can then be forced to cleave leaving pure product to be eluted from the column.
Q Sepharose resin is usually used as an anion exchanging resin that separates proteins based on charge. This method utilizes the special affinity that LYTAG has to quaternary amines, with the controlled cleaving of intein using pH shift and temperature change. Initially, the LYTAG-intein fusion protein is bound to the Q Sepharose column utilizing its specific affinity for quaternary amines rather than ionic interaction. The fusion protein is bound to the column using high salt concentrations to force off other proteins that might have bound to the column with ionic interactions. Equilibrating the column to a lower pH and raising the temperature induces intein self-cleavage. The pure product can then be eluted from the column. From qualitative analysis of SDS-PAGE gel, the purity of the product protein appears to be high.
Because of the ability of Q Sepharose to be scaled up, this technology could mean lab scale research and drug development would more easily translate to industry. It would also be a step toward introducing intein technology to industry. This process could be implemented in the bio-pharmaceutical industry using existing Q Sepharose columns with minimal regulatory approval or change to current setup.
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division - See also TI: Comprehensive Quality by Design in Pharmaceutical Development and Manufacture