An Innovative Method for the Purification of Recombinant Proteins

by Adams, Paul D.; Edwards, Emily Lauren

The purifn. of recombinant proteins is the first step in prepn. for the study of protein structure-function relationship. Affinity tags are put on proteins to help overexpression in sol. forms, and to ease purifn. of recombinant proteins. However, the affinity tag could interfere with protein characterization studies; thus, it must be removed, prior to study. This is accomplished using a protease, such as thrombin, which targets a specific cleavage site between the affinity tag and the recombinant protein of interest. Thrombin, however, remains in soln. with the protein as well. Heparin is known to bind to thrombin and can be used to remove thrombin from the recombinant protein mixt. We are currently testing a method that combines two chromatog. methods, nickel-sepharose that exploits the polyhistidine (His)-tag affinity for nickel, and heparin-sepharose that exploits thrombin's affinity for Heparin, to purify recombinant proteins in one step. We are using the well-studied Ras protein, Cdc42Hs (Cell division cycle 42 Homo sapiens) with an N-terminal His-tag, as our model recombinant protein in our efforts to improve protein purifn. efficiency. Cdc42 was expressed in bacterial cell lines, and protein purifn. was verified through SDS-PAGE. Densiometric anal. compared the amts. of Cdc42Hs purified with the combination column chromatog. procedure to the amts. of protein purified with nickel-sepharose and heparin-sepharose chromatog. sep. The data suggests that combining nickel-sepharose and heparin-sepharose into one column provides a more efficient, and timesaving method to increase recombinant protein prodn.