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.

中文翻译：

---

从大肠杆菌包涵体生产活性N端截短的Ulp1（SUMO蛋白酶1）催化域的新方法。

SUMO融合系统被广泛用于促进重组表达和难以表达的蛋白质的生产。纯化重组融合蛋白后，可通过酵母半胱氨酸蛋白酶SUMO蛋白酶1（Ulp1）去除SUMO标签，该蛋白酶可特异性识别SUMO结构域的三级折叠。目前，催化结构域的残基403-621的表达用于获得可溶性和具有生物活性的Ulp1。然而，我们已经观察到可溶性和催化活性的Ulp1403-621抑制大肠杆菌宿主细胞的生长。在当前的研究中，我们展示了一种从His标记的N末端截短的变体残基416-621产生活性Ulp1催化结构域的替代途径，其在大肠杆菌包涵体中表达并随后重新折叠。表达不溶性Ulp1416-621变体有利于获得更高的产量。从1L高细胞密度大肠杆菌分批发酵培养物中分离的包涵体中回收到约285mg的重组Ulp1416-621。在7.5 M尿素中惰性和变性的Ulp1416-621的Ni2 +亲和纯化后，测试了不同的重折叠条件，这些条件具有不同的l-精氨酸浓度，pH和温度。我们已在室温下成功地将酶在0.25 M精氨酸和0.5 M Tris-HCl（pH 7）中折叠。从1 L高细胞密度大肠杆菌培养物中可以产生约80 mg的活性Ulp1416-621催化域。