Endolysin-based autolytic E. coli system for facile recovery of recombinant proteins
Endolysin-based autolytic E. coli system for facile recovery of recombinant proteins
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基于内溶素的自溶大肠杆菌系统，可轻松回收重组蛋白
DOI：
10.1021/acs.jafc.1c00059
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发表时间：
2021
影响因子：
6.1
通讯作者：
Xia Wu
中科院分区：
文献类型：
--
作者：
Jian Zha;Zhiqiang Liu;Runcong Sun;Guoli Gong;Jonathan S. Dordick;Xia Wu
从大肠杆菌胞质中回收重组蛋白依赖于机械、化学和/或酶方法对细胞的破坏，这通常会导致细胞的不完全破坏或蛋白质变性。可控自溶E。Colistrains被设计用来促进重组蛋白的纯化；然而，这些菌株存在回收率低、细胞裂解缓慢或广泛的菌株工程等问题。在这里，我们报告了一种改进的、高效的可编程自溶E。大肠杆菌平台，在诱导T4溶菌酶的表达和细胞穿透肽的N端融合时启动细胞裂解。通过改造多肽序列和拷贝数，并将融合裂解基因整合到你的体内。在诱导后30分钟内，无论细胞年龄大小，99.97%以上的细胞都能裂解。我们进一步测试了重组溶葡萄球菌酶(LST)的表达和释放，结果表明，在LST表达3h后诱导4h裂解基因，细胞裂解率为98.97%。与冻融法和超声法裂解细胞所得的LST相比，该体系获得的LST产量相同，但活性提高了1.63倍。这种自溶平台显示出在大规模微生物生产蛋白质和其他生物聚合物方面的潜力。
Recovery of recombinant proteins from theEscherichia colicytoplasm depends on cell disruption by mechanical, chemical, and/or enzymatic methods, which usually cause incomplete cell breakage or protein denaturation. Controllable autolyticE. colistrains have been designed to facilitate the purification of recombinant proteins; however, these strains suffer from low recovery yield, slow cell lysis, or extensive strain engineering. Herein, we report an improved, highly efficient programmable autolyticE. coliplatform, in which cell lysis is initiated upon the induced expression of T4 lysozyme with N-terminal fusion of a cell-penetrating peptide. Through the engineering of the peptide sequence and copy number, and by incorporating the fusion lytic gene into theE. coligenome, more than 99.97% of cells could be lysed within 30 min of induction regardless of cell age. We further tested the expression and release of a recombinant enzyme lysostaphin (Lst) and demonstrated that 4 h induction of the lytic gene after 3 h of Lst expression resulted in 98.97% cell lysis. Lst obtained from this system had the same yield, yet 1.63-fold higher activity, compared with that obtained from cells lysed by freeze–thawing and sonication. This autolytic platform shows potential for use in large-scale microbial production of proteins and other biopolymers.