Nikodinovic Jasmina, Priestley Nigel D
Department of Chemistry, University of Montana, Missoula, MT 59812-1656, USA.
Plasmid. 2006 Nov;56(3):223-7. doi: 10.1016/j.plasmid.2006.05.002. Epub 2006 Jun 27.
An Escherichia coli-Streptomyces shuttle vector (pJN100) was constructed, by inserting an origin of transfer (oriT), derived from the E. coli broad host range plasmid RK2, into pANT1202, a high-copy-number vector for gene expression in Streptomyces. The resulting conjugably transferable vector contains the pANT1202-derived SnpR (LysR-like protein) activated snpA promoter that drives strong heterologous expression of proteins. We initially demonstrated that plasmid pJN100 was transferred with high frequency (10(-5-7) exconjugants per recipient) into several Streptomyces strains that were refractory to transformation by other means. Plasmid pJN100 was also shown to be stable in E. coli and Streptomyces. We confirmed functional protein expression by using a pJN100 derivative to complement a mutant of Streptomyces griseus with a disrupted chromosomal copy of the gene nonM, a gene encoding an essential reductase in the nonactin biosynthesis gene cluster. High levels of protein expression were confirmed using Western blotting to assess the production of the serine esterase NonR, an enzyme responsible for nonactin resistance in the nonactin producer S. griseus.
构建了一种大肠杆菌-链霉菌穿梭载体（pJN100），方法是将源自大肠杆菌广宿主范围质粒RK2的转移起始位点（oriT）插入pANT1202，pANT1202是一种用于链霉菌中基因表达的高拷贝数载体。所得的可接合转移载体含有源自pANT1202的SnpR（类LysR蛋白）激活的snpA启动子，该启动子驱动蛋白质的强异源表达。我们最初证明质粒pJN100能够以高频率（每受体10^(-5 - 7)个接合后体）转移到几种难以通过其他方法转化的链霉菌菌株中。质粒pJN100在大肠杆菌和链霉菌中也表现出稳定性。我们通过使用pJN100衍生物互补灰色链霉菌的一个突变体来证实功能性蛋白质表达，该突变体的染色体拷贝中的nonM基因被破坏，nonM基因是一种在诺卡菌素生物合成基因簇中编码必需还原酶的基因。使用蛋白质印迹法评估丝氨酸酯酶NonR的产生，证实了高水平的蛋白质表达，NonR是诺卡菌素产生菌灰色链霉菌中负责诺卡菌素抗性的一种酶。
