A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies.

The SUMO fusion system is widely used to facilitate recombinant expression and production of difficult-to-express proteins. After purification of the recombinant fusion protein, removal of the SUMO-tag is accomplished by the yeast cysteine protease, SUMO protease 1 (Ulp1), which specifically recognizes the tertiary fold of the SUMO domain. At present, the expression of the catalytic domain, residues 403-621, is used for obtaining soluble and biologically active Ulp1. However, we have observed that the soluble and catalytically active Ulp1403-621 inhibits the growth of E. coli host cells. In the current study, we demonstrate an alternative route for producing active Ulp1 catalytic domain from a His-tagged N-terminally truncated variant, residues 416-621, which is expressed in E. coli inclusion bodies and subsequently refolded. Expressing the insoluble Ulp1416-621 variant is advantageous for achieving higher production yields. Approximately 285 mg of recombinant Ulp1416-621 was recovered from inclusion bodies isolated from 1 L of high cell-density E. coli batch fermentation culture. After Ni2+-affinity purification of inactive and denatured Ulp1416-621 in 7.5 M urea, different refolding conditions with varying l-arginine concentration, pH, and temperature were tested. We have successfully refolded the enzyme in 0.25 M l-arginine and 0.5 M Tris-HCl (pH 7) at room temperature. Approximately 80 mg of active Ulp1416-621 catalytic domain can be produced from 1 L of high cell-density E. coli culture. We discuss the applicability of inclusion body-directed expression and considerations for obtaining high expression yields and efficient refolding conditions to reconstitute the active protein fold.