Development of anaerobically inducible nar promoter expression vectors for the expression of recombinant proteins in Escherichia coli.
Dissolved oxygen (DO)-controlled nar promoter expression vectors were constructed, and their expression efficiency was compared with that of the T7 promoter pET22 expression vector by expressing human growth hormone (hGH), enhanced green fluorescence protein (EGFP), and β-tyrosinase in Escherichia coli cells. The nar promoter expression vector pRBS, which was engineered with a 5'-untranslated region and ribosomal binding site for the T7 promoter, expressed hGH at a rate of up to 32% of the total cellular proteins (TCP) in E. coli W3110narL⁻. The expression level of hGH was further enhanced, up to ~42% of the TCP, by adding the N-terminal peptide tag of β-galactosidase to hGH, which was comparable to the expression of ~43% of the TCP in pET-lac:hGH/BL21(DE3). A further engineered expression vector, pRBS(fnr), which coexpressed fumarate/nitrate reductase (fnr), expressed more EGFP than pET22 in BL21(DE3). In addition, recombinant β-tyrosinase was successfully expressed at a rate of up to ~45% of the TCP in pRBS(fnr) in W3110narL⁻. From these results, the DO-controlled nar promoter system developed in this study can be considered a reliable and cost-effective expression system for protein production, especially in large-scale fermentation, as an alternative to the pET/BL(DE3) system.
Department of Chemical and Biomolecular Engineering (BK21 Program), Bioinformatics Research Center, BioProcess Engineering Research Center and Center for Ultramicrochemical Process Systems, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea.
Nag-Jong
NJ
Kim
Jong Hyun
JH
Choi
Yeon Chul
YC
Kim
Jongwon
J
Lee
Sang Yup
SY
Lee
Ho Nam
HN
Chang
Pyung Cheon
PC
Lee
D000693:Anaerobiosis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014447:Tyrosine Phenol-Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D019382:Human Growth Hormone; D049452:Green Fluorescent Proteins; D014447:Tyrosine Phenol-Lyase