Alpert C A, Chassy B M
Laboratory of Microbiology and Immunology, National Institute of Dental Research, Bethesda, MD 20892.
Gene. 1988;62(2):277-88. doi: 10.1016/0378-1119(88)90565-3.
The lactose-specific factor III (FIIIlac of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was isolated from Lactobacillus casei and purified to homogeneity by conventional protein purification methods. Its apparent native Mr, estimated from steric exclusion chromatography (approx. 39 kDa), and subunit Mr, estimated from sodium dodecyl sulfate-polyacrylamide gels, indicated that it exists as a trimer of identical subunits of 13 kDa. The gene for FIII L. casei lac was cloned into Escherichia coli using the vector pUC18. The coding sequences were contained on an 860-bp BglII-HindIII DNA fragment of the L. casei lactose plasmid, pLZ64. A protein identical in properties to FIII L. casei lac was isolated from clones of E. coli carrying this DNA insert. The nucleotide sequence of the FIII L. casei lac gene was determined by the dideoxy chain-termination technique. The 336-bp open reading frame for FIII L. casei lac was followed by a stem-loop structure, analogous to a Rho-independent terminator. We concluded that the FIII L. casei lac was the terminal gene in what appears to be an operon comprised of the lactose-PTS-P-beta Gal-coding genes. Comparison of the deduced amino acid sequence of FIII L. caseilac with the amino acid sequence of FIII S. aureus lac (derived from peptide sequencing) demonstrated a high degree of homology (49 identical residues and 21 conservative exchanges out of 103 total aa residues). The FIII L. casei lac lacked his82, previously identified as the phosphorylation site of FIII S. aureus. lac His80 was proposed to be the site of histidyl phosphorylation of FIII L. casei lac.
从干酪乳杆菌中分离出磷酸烯醇丙酮酸依赖性磷酸转移酶系统（PTS）的乳糖特异性因子III（FIIIlac），并通过常规蛋白质纯化方法将其纯化至同质。通过空间排阻色谱法估计其表观天然Mr（约39 kDa），以及通过十二烷基硫酸钠-聚丙烯酰胺凝胶估计其亚基Mr，表明它以13 kDa相同亚基的三聚体形式存在。使用载体pUC18将干酪乳杆菌FIII lac的基因克隆到大肠杆菌中。编码序列包含在干酪乳杆菌乳糖质粒pLZ64的860 bp BglII-HindIII DNA片段上。从携带该DNA插入片段的大肠杆菌克隆中分离出一种性质与干酪乳杆菌FIII lac相同的蛋白质。通过双脱氧链终止技术确定了干酪乳杆菌FIII lac基因的核苷酸序列。干酪乳杆菌FIII lac的336 bp开放阅读框后面是一个茎环结构，类似于不依赖Rho的终止子。我们得出结论，干酪乳杆菌FIII lac是一个似乎由乳糖-PTS-P-β-半乳糖苷酶编码基因组成的操纵子中的末端基因。将干酪乳杆菌FIII lac推导的氨基酸序列与金黄色葡萄球菌FIII lac的氨基酸序列（来自肽测序）进行比较，发现高度同源（103个总氨基酸残基中有49个相同残基和21个保守交换）。干酪乳杆菌FIII lac缺乏his82，以前被确定为金黄色葡萄球菌FIII的磷酸化位点。lac His80被认为是干酪乳杆菌FIII lac的组氨酸磷酸化位点。
