Solaiman D K, Somkuti G A
U.S. Department of Agriculture, Agricultural Research Service, Philadelphia, PA 19118, USA.
Appl Microbiol Biotechnol. 1995 May-Jun;43(2):285-90. doi: 10.1007/BF00172826.
A 63-base-pair synthetic promoter, sP1, was synthesized on the basis of the nucleotide sequence of a putative Streptococcus thermophilus promoter. When inserted upstream from the Streptomyces cho operon in a recombinant plasmid, pUCO195P-36, sP1 activated the expression of the cho genes in Escherichia coli, as shown by the production of cholesterol oxidase by the transformants. The sP1-driven cholesterol oxidase production in pUCO195P-36-transformed cells was estimated to be 40% of that produced by P(lac)-mediated cho expression in a pUCO193-containing host. The recombinant pUCO195P-36 appeared to be segregationally less stable in E. coli DH5 alpha than in HB101. Its non-expressing counterpart, pUCO195P-1, was stable in both E. coli strains. The activity of sP1 was further demonstrated in E. coli by the expression of a Streptomyces melC operon. When placed upstream from the test operon in the pMCU22aPa construct, sP1 activated the melC expression as shown by the production of tyrosinase at (3.0 +/- 0.3) x 10(-3) U/mg and (16.0 +/- 1.0) x 10(-3) U/mg protein equivalent of cell extract in the absence and presence of isopropyl beta-D-thiogalactopyranoside, respectively. The presence of a counter-oriented P(lac) at the 3' end of the operon in the pMCU22bPa plasmid reduced the sP1-mediated tyrosinase production by about 85%.
一个63个碱基对的合成启动子sP1，是根据嗜热链球菌假定启动子的核苷酸序列合成的。当将其插入重组质粒pUCO195P - 36中链霉菌cho操纵子的上游时，sP1激活了大肠杆菌中cho基因的表达，转化体产生胆固醇氧化酶就表明了这一点。据估计，在pUCO195P - 36转化的细胞中，sP1驱动产生的胆固醇氧化酶量是含pUCO193的宿主中由P(lac)介导的cho表达产生量的40%。重组体pUCO195P - 36在大肠杆菌DH5α中似乎比在HB101中分离稳定性更低。其不表达的对应物pUCO195P - 1在两种大肠杆菌菌株中都很稳定。通过链霉菌melC操纵子的表达，进一步在大肠杆菌中证明了sP1的活性。当置于pMCU22aPa构建体中测试操纵子的上游时，sP1激活了melC的表达，在不存在和存在异丙基β - D - 硫代半乳糖苷的情况下，细胞提取物中酪氨酸酶的产生量分别为(3.0±0.3)×(10^{-3}) U/mg和(16.0±1.0)×(10^{-3}) U/mg蛋白质当量。pMCU22bPa质粒中操纵子3'端反向的P(lac)的存在使sP1介导的酪氨酸酶产生量降低了约85%。
