Abstract

Mature lysostaphin (mLst) is a glycineglycine endopeptidase, capable of specifically cleaving penta-glycine crosslinker in the peptidoglycan of Staphylococcus aureus cell wall. It is a very effective therapeutic enzyme to kill the multidrug-resistant S. aureus often encountered in hospital acquired infections. Fusing cellulose binding domain (CBD) to mLst significantly reduced the insoluble expression of mLst in E. coli. Employing mLst-cleavable peptides as fusion linkers leaded to an effective self-cleavage expression that CBD and mLst could be completely cleaved off from the fusions during the expression process. The presence of residue linker fragment at N-terminus of the cleaved-off mLst strongly inhibited the cell lytic activity of the recovered recombinant mLst, and only ~ 50% of the wild-type mLst activity could be retained. Intact CBD-Lst fusions were obtained when uncleavable peptide linkers were employed. With CBD at N-terminus of mLst, the intact fusion completely lost its cell lytic activity but the dipeptidase activity still remained. In contrast, approximately 10% cell lytic activity of mLst still could be maintained for the fusion with CBD at C-terminus of mLst.

Key points

• CBD fusion enhanced soluble expression of recombinant lysostaphin.

• In vivo self-cleavage of fusion linkers by the expressed lysostaphin fusions.

• Self-cleaved lysostaphin fusions retain most of dipeptidase but lose 50% cell lytic activity.

中文翻译：

---

重组溶葡萄球菌素的纤维素结合域融合自切割表达

摘要

成熟溶葡萄球菌素 (mLst) 是一种甘氨酸甘氨酸内肽酶，能够特异性切割金黄色葡萄球菌细胞壁肽聚糖中的五甘氨酸交联剂。它是一种非常有效的治疗酶，可以杀死医院获得性感染中经常遇到的多重耐药金黄色葡萄球菌。将纤维素结合域 (CBD) 与 mLst 融合显着降低了 mLst 在大肠杆菌中的不溶性表达. 使用 mLst 可切割肽作为融合接头导致有效的自切割表达，在表达过程中 CBD 和 mLst 可以从融合中完全切割下来。切割下的 mLst N 端残基接头片段的存在强烈抑制了回收的重组 mLst 的细胞裂解活性，并且仅能保留约 50% 的野生型 mLst 活性。当使用不可切割的肽接头时，获得了完整的 CBD-Lst 融合。由于 CBD 在 mLst 的 N 端，完整的融合完全失去了其细胞溶解活性，但二肽酶活性仍然存在。相比之下，mLst 的大约 10% 的细胞溶解活性仍然可以保持，以便在 mLst 的 C 端与 CBD 融合。

关键点

• CBD 融合增强了重组溶葡萄球菌素的可溶性表达。

• 表达的溶葡萄球菌素融合体对融合接头的体内自切割。

• 自切割溶葡萄球菌素融合蛋白保留了大部分二肽酶，但失去了 50% 的细胞裂解活性。