Use of ribosomal promoters from Burkholderia cenocepacia and Burkholderia cepacia for improved expression of transporter protein in Escherichia coli.
Expression of heterologous protein in Escherichia coli usually based on the IPTG-inducible expression systems. The use of these systems for membrane protein production, however, usually caused cytotoxic problem that affected the yield and functional characterization of the protein. Optimization of these systems for transporter protein production is time-consuming and is usually ineffective. Here, we described the use of the ribosomal promoters P(s12) from Burkholderia cenocepacia LMG16656 and from Burkholderia cepacia MBA4 for efficient expression of functional transporter protein in E. coli. These promoters were used to drive the expression of a transmembrane protein, Deh4p, which help transport monohaloacetates into B. cepacia MBA4 for metabolism. Production of Deh4p in E. coli using an IPTG-inducible promoter resulted in no expression in uninduced condition and cell lysis in the presence of IPTG. Moreover, it has been reported that IPTG increased the endogenous production of other permeases such as LacZ and MelB. Cells expressing Deh4p from a P(s12) promoter grew normally in rich medium and which did not increase the expression of other permease. Uptake of (14)C-monochloroacetic acid has confirmed the production of the transporter protein in these cells. The results showed that the constitutive ribosomal protein promoters from the Burkholderia sp. could be used for effective expression of transporter protein in E. coli without causing any detrimental and unnecessary effect.
Yu
Manda
M
Molecular Microbiology Laboratory, Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China.
Tsang
Jimmy S H
JS
D001426:Bacterial Proteins / Q000235:genetics*; D016956:Burkholderia cepacia / Q000235:genetics*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D015870:Gene Expression; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012269:Ribosomal Proteins / Q000235:genetics*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D011994:Recombinant Proteins; D012269:Ribosomal Proteins