title: Construction of heat-inducible expression vector of Corynebacterium glutamicum and C. ammoniagenes: fusion of lambda operator with promoters isolated from C. ammoniagenes.
abstract: The heat-inducible expression vectors for Corynebacterium glutamicum and C. ammoniagenes were constructed by using the lambdaOL1 and the cryptic promoters, CJ1 and CJ4 that express genes constitutively in C. ammoniagenes.. Although the promoters were isolated from C. ammoniagenes, CJ1 and CJ4 were also active in C. glutamicum. To construct vectors, the OL1 from the lambdaPL promoter was isolated and fused to the CJ1 and CJ4 promoters by recombinant PCR. The resulting artificial promoters, CJ1O and CJ4O, which have one lambdaOL1, and CJ1OX2, which has two successive lambdaOL1, were fused to the green fluorescent protein (GFP) gene followed by subcloning into pCES208. The expression of GFP in the corynebacteria harboring the vectors was regulated successfully by the temperature sensitive cI857 repressor. Among them, C. ammoniagenes harboring plasmid pCJ1OX2G containing GFP fused to CJ1OX2 showed more GFP than the other ones and the expression was tightly regulated by the repressor. To construct the generally applicable expression vector using the plasmid pCJ1OX2G, the His-tag, enterokinase (EK) moiety, and the MCS were inserted in front of the GFP gene. Using the vector, the expression of pyrR from C. glutamicum was tried by temperature shift-up. The results indicated that the constructed vectors (pCeHEMG) can be successfully used in the expression and regulation of foreign genes in corynebacteria.
authors.affiliation: Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin 449-791, Korea.
authors.forename: Jong-Uk
authors.initials: JU
authors.lastname: Park
authors.forename: Jae-Hyung
authors.initials: JH
authors.lastname: Jo
authors.forename: Young-Ji
authors.initials: YJ
authors.lastname: Kim
authors.forename: So-Sun
authors.initials: SS
authors.lastname: Chung
authors.forename: Jin-Ho
authors.initials: JH
authors.lastname: Lee
authors.forename: Hyune Hwan
authors.initials: HH
authors.lastname: Lee
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins