Siehnel R J, Worobec E A, Hancock R E
Department of Microbiology, University of British Columbia, Vancouver, Canada.
J Bacteriol. 1988 May;170(5):2312-8. doi: 10.1128/jb.170.5.2312-2318.1988.
The gene encoding the outer membrane phosphate-selective porin protein P from Pseudomonas aeruginosa was cloned into Escherichia coli. The protein product was expressed and transported to the outer membrane of an E. coli phoE mutant and assembled into functional trimers. Expression of a product of the correct molecular weight was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, using polyclonal antibodies to protein P monomer and trimer forms. Protein P trimers were partially purified from the E. coli clone and shown to form channels with the same conductance as those formed by protein P from P. aeruginosa. The location and orientation of the protein P-encoding (oprP) gene on the cloned DNA was identified by three methods: (i) mapping the insertion point of transposon Tn501 in a previously isolated P. aeruginosa protein P-deficient mutant; (ii) hybridization of restriction fragments from the cloned DNA to an oligonucleotide pool synthesized on the basis of the amino-terminal protein sequence of protein P; and (iii) fusion of a PstI fragment of the cloned DNA to the amino terminus of the beta-galactosidase gene of pUC8, producing a fusion protein that contained protein P-antigenic epitopes. Structural analysis of the cloned DNA and P. aeruginosa chromosomal DNA revealed the presence of two adjacent PstI fragments which cross-hybridized, suggesting a possible gene duplication. The P-related (PR) region hybridized to the oligonucleotide pool described above. When the PstI fragment which contained the PR region was fused to the beta-galactosidase gene of pUC8, a fusion protein was produced which reacted with a protein P-specific antiserum. However, the restriction endonuclease patterns of the PR region and the oprP gene differed significantly beyond the amino-terminal one-third of the two genes.
将铜绿假单胞菌编码外膜磷酸盐选择性孔蛋白P的基因克隆到大肠杆菌中。该蛋白产物得以表达，并转运至大肠杆菌phoE突变体的外膜，组装成功能性三聚体。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和蛋白质印迹（免疫印迹）分析，使用针对蛋白P单体和三聚体形式的多克隆抗体，证实了正确分子量产物的表达。从大肠杆菌克隆体中部分纯化出蛋白P三聚体，结果表明其形成的通道与铜绿假单胞菌的蛋白P形成的通道具有相同的电导率。通过三种方法确定了克隆DNA上编码蛋白P（oprP）基因的位置和方向：（i）绘制转座子Tn501在先前分离的铜绿假单胞菌蛋白P缺陷突变体中的插入点；（ii）将克隆DNA的限制性片段与基于蛋白P的氨基末端蛋白序列合成的寡核苷酸池杂交；（iii）将克隆DNA的PstI片段与pUC8的β-半乳糖苷酶基因的氨基末端融合，产生一种包含蛋白P抗原表位的融合蛋白。对克隆DNA和铜绿假单胞菌染色体DNA的结构分析显示，存在两个相邻的相互交叉杂交的PstI片段，表明可能存在基因重复。P相关（PR）区域与上述寡核苷酸池杂交。当包含PR区域的PstI片段与pUC8的β-半乳糖苷酶基因融合时，产生了一种与蛋白P特异性抗血清发生反应的融合蛋白。然而，PR区域和oprP基因的限制性内切酶图谱在两个基因的氨基末端三分之一以外有显著差异。
