Biomedical subjects

Sung Kuk Lee

Publications and source records attributed to Sung Kuk Lee.

6 recordsLinked to original sources

PubMed2006

A tightly regulated inducible expression system utilizing the fim inversion recombination switch.

The fim inversion system of Escherichia coli (E. coli) can behave as a unidirectional switch in an efficient manner. We have developed a new expression system for E. coli, comprising the arabinose-inducible fimE gene and the fim invertible DNA segment containing a constitutively active promoter. In this system, the target gene is cloned with the promoter in the OFF orientation, resulting in no transcribed product. When induced by arabinose, the active promoter is switched to the ON orientation via FimE-catalyzed DNA inversion, and the gene is expressed. Our expression system exhibited very tightly controlled basal expression and high induced expression, with simple induction by inexpensive arabinose. These characteristics make our system suitable for large-scale expression or for production of toxic proteins.

Chromosome Inversion↗

PubMed2006

A Salmonella-based, propionate-inducible, expression system for Salmonella enterica.

The expression and regulatory properties of a propionate-regulated overexpression system (Salmonella enterica prpBCDE promoter (P(prpB)) and positive regulator (prpR)) were evaluated in wild-type S. enterica serovar Typhimurium TR6583 and prpB(-) or prpD(-) versions of this strain and compared with the arabinose-regulated T7 expression system. The wild-type strain showed low expression in the absence of propionate and high expression in the presence of propionate under all growth conditions. In 96-well plates and culture tubes, the wild-type strain exhibited a long delay before full induction; the time delay was significantly shorter in shake flasks. The prpD(-) strain exhibited low expression in the presence of glucose, highly regulatable expression over a wide range of propionate concentrations, and, in contrast to the wild-type strain, fast induction to full expression under all growth conditions. In contrast, the prpB(-) strain showed very high background expression in both culture tubes and shake flasks.

Arabinose↗

PubMed2006

Propionate-regulated high-yield protein production in Escherichia coli.

A new expression system containing the Salmonella enterica prpBCDE promoter (P(prpB)) responsible for expression of the propionate catabolic genes (prp BCDE) and prpR encoding the positive regulator of this promoter has been developed and tested. The main features of the expression system compared to those based on the bacteriophage T7 promoter are low background expression and high induced expression in Escherichia coli strains BL21, BL21(DE3), MG1655, and W3110. In addition, propionate is an inexpensive, simple-to-use, nontoxic inducer that is attractive for large-scale protein production. Hence, this new system is highly complementary to the widely used T7 promoter-driven expression systems.

Bacteriophage T7↗

PubMed2005

A propionate-inducible expression system for enteric bacteria.

A series of new expression vectors (pPro) have been constructed for the regulated expression of genes in Escherichia coli. The pPro vectors contain the prpBCDE promoter (P(prpB)) responsible for expression of the propionate catabolic genes (prpBCDE) and prpR encoding the positive regulator of this promoter. The efficiency and regulatory properties of the prpR-P(prpB) system were measured by placing the gene encoding the green fluorescent protein (gfp) under the control of the inducible P(prpB) of E. coli. This system provides homogenous expression in individual cells, highly regulatable expression over a wide range of propionate concentrations, and strong expression (maximal 1,500-fold induction) at high propionate concentrations. Since the prpBCDE promoter has CAP-dependent activation, the prpR-P(prpB) system exhibited negligible basal expression by addition of glucose to the medium.

Bacterial Proteins↗

PubMed2005

Catabolite repression of the propionate catabolic genes in Escherichia coli and Salmonella enterica: evidence for involvement of the cyclic AMP receptor protein.

Previous studies with Salmonella enterica serovar Typhimurium LT2 demonstrated that transcriptional activation of the prpBCDE operon requires the function of transcription factor PrpR, sigma-54, and IHF. In this study, we found that transcription from the prpBCDE and prpR promoters was down-regulated by the addition of glucose or glycerol, indicating that these genes may be regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. Targeted mutagenesis of a putative CRP-binding site in the promoter region between prpR and prpBCDE suggested that these genes are under the control of CRP. Furthermore, cells with defects in cya or crp exhibited reduced transcriptional activation of prpR and prpBCDE in Escherichia coli. These results demonstrate that propionate metabolism is subject to catabolite repression by the global transcriptional regulator CRP and that this regulation is effected through control of both the regulator gene prpR and the prpBCDE operon itself. The unique properties of the regulation of these two divergent promoters may have important implications for mechanisms of CRP-dependent catabolite repression acting in conjunction with a member of the sigma-54 family of transcriptional activators.

Escherichia coli↗

PubMedDate not supplied

Effect of glucose or glycerol as the sole carbon source on gene expression from the Salmonella prpBCDE promoter in Escherichia coli.

We have developed an expression system (Salmonella-based pPro system) containing the Salmonella enterica prpBCDE promoter (PprpB) and prpR encoding the positive transcriptional regulator of this promoter. In this study, the transcriptional efficiency of the pPro expression system was measured by placing the gene encoding the green fluorescent protein (gfp) under the control of PprpB and growing cells containing this construct in minimal medium supplemented with glucose or glycerol as a sole carbon source. In wild-type Escherichia coli (E. coli) BL21, the system exhibited high induced expression as well as high background expression; however, in E. coli JSB, a sbm-ygfDGHI deletion mutant of E. coli BL21(DE3), the system showed low background expression and high induced expression. The system exhibited homogeneous expression at the single-cell level, highly regulatable expression over a wide range of propionate concentrations, and fully induced expression at a low propionate concentration relative to that needed to induce the system in rich, undefined medium. The expression system is comparable to the widely used T7 promoter-driven expression systems in glucose or glycerol minimal medium.

Carbon↗