Oudega B, Clark D, Stegehuis F, Majoor M J, Luirink J
Department of Molecular Microbiology, Faculty of Biology, Free University, Amsterdam, The Netherlands.
FEMS Microbiol Lett. 1993 Apr 15;108(3):353-9. doi: 10.1111/j.1574-6968.1993.tb06127.x.
By genetic exchange and in vitro mutagenesis a hybrid beta-lactamase was constructed that contained the pCloDF13-encoded bacteriocin release protein signal peptide plus a cysteine residue coupled to the mature portion of beta-lactamase. Immunoblotting, labelling with [3H]palmitate in the presence and absence of globomycin, and pulse-chase experiments revealed that this hybrid construct is modified with lipid and processed into a lipid-modified beta-lactamase. Subcellular localization studies revealed that this hybrid is localized both in the cytoplasmic and outer membranes of Escherichia coli cells. A mutant derivative with an incomplete lipobox (LVG instead of LVAC+1) was not processed and was found in the cytoplasmic membranes.
通过基因交换和体外诱变构建了一种杂合β-内酰胺酶，它含有pCloDF13编码的细菌素释放蛋白信号肽以及与β-内酰胺酶成熟部分相连的一个半胱氨酸残基。免疫印迹、在有无球霉素存在的情况下用[3H]棕榈酸酯标记以及脉冲追踪实验表明，这种杂合构建体被脂质修饰并加工成脂质修饰的β-内酰胺酶。亚细胞定位研究表明，这种杂合体定位于大肠杆菌细胞的细胞质膜和外膜。一种具有不完全脂质盒（LVG而不是LVAC +1）的突变衍生物未被加工，而是存在于细胞质膜中。
