Fu Linglong, Sun Mengning, Wen Weizhang, Dong Na, Li Defa
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, People's Republic of China.
Appl Microbiol Biotechnol. 2022 Dec;106(23):7805-7817. doi: 10.1007/s00253-022-12235-z. Epub 2022 Oct 19.
Small ubiquitin-like modifier (SUMO) tag is widely used to promote soluble expression of exogenous proteins, which can then be cleaved by ubiquitin-like protease 1 (Ulp1) to obtain interested protein. But the application of Ulp1 in large-scale recombinant protein production is limited by complicated purification procedures and high cost. In this study, we describe an efficient and simple method of extracellular production of Ulp1 using a leaky Escherichia coli BL21(DE3), engineered by deleting the peptidoglycan-associated outer membrane lipoprotein (pal) gene. Ulp1 was successfully leaked into extracellular supernatant by the BL21(DE3)-Δpal strain after IPTG induction. The addition of 1% glycine increased the extracellular production of Ulp1 approximately four fold. Moreover, extracellular Ulp1 without purification had high activities for cleaving SUMO fusion proteins, and antimicrobial peptide pBD2 obtained after cleavage can inhibit the growth of Staphylococcus aureus. The specific activity of extracellular Ulp1 containing 1 mM EDTA and 8 mM DTT reached 2.0 × 10 U/L. Another commonly used protease, human rhinovirus 3C protease, was also successfully secreted by leaky E. coli strains. In conclusion, extracellular production of tool enzymes is an attractive way for producing large-scale active recombinant proteins at a lower cost for pharmaceutical, industrial, and biotechnological applications. KEY POINTS: • First report of extracellular production of Ulp1 in leaky Escherichia coli BL21(DE3) strain. • One percent glycine addition into cultivation medium increased the extracellular production of Ulp1 approximately four fold. • The specific activity of extracellular Ulp1 produced in this study reached 2.0 × 10 U/L.
小泛素样修饰物（SUMO）标签被广泛用于促进外源蛋白的可溶性表达，随后可被泛素样蛋白酶1（Ulp1）切割以获得感兴趣的蛋白。但Ulp1在大规模重组蛋白生产中的应用受到复杂的纯化程序和高成本的限制。在本研究中，我们描述了一种利用缺失肽聚糖相关外膜脂蛋白（pal）基因构建的渗漏型大肠杆菌BL21(DE3)高效且简单的胞外生产Ulp1的方法。IPTG诱导后，Ulp1通过BL21(DE3)-Δpal菌株成功渗漏到胞外上清液中。添加1%甘氨酸使Ulp1的胞外产量增加了约四倍。此外，未经纯化的胞外Ulp1对切割SUMO融合蛋白具有高活性，切割后获得的抗菌肽pBD2可抑制金黄色葡萄球菌的生长。含有1 mM EDTA和8 mM DTT的胞外Ulp1的比活性达到2.0×10 U/L。另一种常用的蛋白酶人鼻病毒3C蛋白酶也通过渗漏型大肠杆菌菌株成功分泌。总之，工具酶的胞外生产是一种有吸引力的方式，可在制药、工业和生物技术应用中以较低成本大规模生产活性重组蛋白。要点：•首次报道在渗漏型大肠杆菌BL21(DE3)菌株中胞外生产Ulp1。•向培养基中添加1%甘氨酸使Ulp1的胞外产量增加了约四倍。•本研究中生产的胞外Ulp1的比活性达到2.0×10 U/L。
