The expression of proUK in Escherichia coli: the vgb promoter replaces IPTG and coexpression of argU compensates for rare codons in a hypoxic induction model.
The expression of the proUK gene was improved by the coexpression of the argU gene cloned in a moderate copy number vector. As the proUK gene contains 2% AGG/AGA codons, which is much higher than the normal frequency in E. coli, about 0.14%-0.21%, the argU gene cloned in a multicopy plasmid was coexpressed with the proUK expression vector in our experiments. In E. coli strain BL21(DE3), IPTG is known to induce the expression of T7 RNA polymerase gene and this enzyme can transcribe the proUK gene under the control of the T7 promoter leading to expression of proUK. To replace IPTG by a cheaper alternative on a large scale, we constructed a plasmid in which the vgb promoter--which is known to be activated by the onset of hypoxic conditions--controls the T7RNA polymerase gene expression. Low oxygen conditions were then used to activate the vgb promoter causing T7RNA polymerase gene expression and finally leading to the expression of proUK as inactive inclusion bodies. Our experiments on a large scale in a bioreactor show that the expression of proUK accounts for about 30% of total protein after about 6 h of anaerobic cultivation, so the presented model represents an economical alternative to IPTG induction.
Jiang
L
L
Shanghai Research Center of Biotechnology, Chinese Academy of Sciences, P.R.China. lanjiang@hotmail.com
Yang
Y
Y
Chatterjee
S
S
Seidel
B
B
Wolf
G
G
Yang
S
S
D001426:Bacterial Proteins / Q000235:genetics*; D003062:Codon; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000235:genetics*; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic; D006420:Hemeproteins / Q000235:genetics*; D000860:Hypoxia; D007544:Isopropyl Thiogalactoside; D010101:Oxygen Consumption / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012347:RNA, Transfer, Arg / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics*; D014764:Viral Proteins
D001426:Bacterial Proteins; D003062:Codon; D004792:Enzyme Precursors; D006420:Hemeproteins; D012347:RNA, Transfer, Arg; D011994:Recombinant Proteins; C100771:Vgb protein, Bacteria; D014764:Viral Proteins; D007544:Isopropyl Thiogalactoside; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase