Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 166, Issue -, Pages -Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2019.105507
Keywords
Ubiquitin-like protease (Ulp); Small ubiquitin-like modifier (SUMO); SUMO-Specific protease; Inclusion bodies; Refolding; L-arginine
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Funding
- Fight for Sight Denmark foundation
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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 Ulp1(403-621) 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 Ulp1(416-621) variant is advantageous for achieving higher production yields. Approximately 285 mg of recombinant Ulp1(416-621) 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 Ulp1(416-621) 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 Ulp1(415-621) 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.
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