Rondahl H, Nilsson B, Holmgren E
Department of Microbiology, Kabi Pharmacia, KabiGen, Stockholm, Sweden.
J Biotechnol. 1992 Sep;25(3):269-87. doi: 10.1016/0168-1656(92)90161-2.
A novel gene fusion system has been constructed for fusions to the 5' end of gene zz, encoding a two-domain analogue of staphylococcal protein A designated ZZ. Four different genes were fused to the 5' end of zz, and their gene products were analyzed. One of the genes encodes a protein located intracellularly in Escherichia coli and the other three genes encode gene products destined for secretion across the cytoplasmic membrane by the presence of an amino terminal signal sequence. After production in E. coli, the fusion proteins were purified in a single step by IgG-affinity chromatography. The purified ZZ fusions could be used directly for amino terminal sequencing to confirm the start of translation of the intracellular product and the processing of the signal peptide of the translocated products. This is the first example of ZZ fusions to the C-terminus of gene products. To simplify the general use of fusions to the 5' end of zz, a new plasmid vector was constructed containing a multi restriction enzyme cloning linker and the lacZ' gene which enables screening for production in alpha-complementing supE strains of E. coli on indicator plates.
构建了一种新型基因融合系统，用于与基因zz的5'端融合，该基因编码一种名为ZZ的葡萄球菌蛋白A的双结构域类似物。将四个不同的基因与zz的5'端融合，并对其基因产物进行分析。其中一个基因编码位于大肠杆菌细胞内的一种蛋白质，另外三个基因编码的基因产物因存在氨基末端信号序列而注定要穿过细胞质膜分泌。在大肠杆菌中产生后，融合蛋白通过IgG亲和层析一步纯化。纯化的ZZ融合蛋白可直接用于氨基末端测序，以确认细胞内产物翻译的起始以及转运产物信号肽的加工。这是ZZ与基因产物C末端融合的首个实例。为简化与zz 5'端融合的普遍应用，构建了一种新的质粒载体，其含有多限制酶克隆接头和lacZ'基因，该基因能够在指示平板上筛选在大肠杆菌α互补supE菌株中的表达。
