Mermet-Bouvier P, Chauvat F
Département de Biologie Cellulaire et Moléculaire, Commissariat à l'Energie Atomique, Gif Sur Yvette, France.
Curr Microbiol. 1994 Mar;28(3):145-8. doi: 10.1007/BF01571055.
An expression vector, pFC1, has been constructed based on the promiscuous plasmid RSF1010, which provides autonomous replication in several cyanobacteria of the genera Synechocystis and Synechococcus [Mermet-Bouvier et al., Curr Microbiol 26:323-327]. pFC1 harbors the lambda cI857 repressor-encoding gene and pR promoter, followed by the lambda cro ribosome-binding site and ATG translation initiation codon. The latter is located within the unique NdeI restriction site (CATATG) of pFC1 and can be exposed after cleavage with this enzyme for in-frame fusion with the protein-coding sequence to be expressed. The Escherichia coli lacZ reporter gene cloned in pFC1 appeared to be highly expressed in heat-induced E. coli or cyanobacterial cells. In every case, beta-galactosidase amounted to at least 10% of soluble proteins.
基于可在集胞藻属和聚球藻属的几种蓝细菌中自主复制的多宿主质粒RSF1010构建了表达载体pFC1[默梅特 - 布维耶等人,《当前微生物学》26:323 - 327]。pFC1含有λ cI857阻遏蛋白编码基因和pR启动子,随后是λ cro核糖体结合位点和ATG翻译起始密码子。后者位于pFC1独特的NdeI限制位点(CATATG)内,用该酶切割后可暴露出来,以便与要表达的蛋白质编码序列进行读码框内融合。克隆在pFC1中的大肠杆菌lacZ报告基因在热诱导的大肠杆菌或蓝细菌细胞中似乎高度表达。在每种情况下,β - 半乳糖苷酶至少占可溶性蛋白的10%。