• Journal of Microbiology and Biotechnology
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• v.21 no.3
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• pp.252-255
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• 2011

Both Escherichia coli (E. coli) and Salmonella enterica serovar Typhimurium (S. Typhimurium) have a tdc operon that encodes enzymes involved in a metabolic pathway for the degradation of L-serine and L-threonine. However, S. Typhimurium does not have the tdcR gene, which is a positive regulator in E. coli. In the present study, transcriptional analysis revealed that tdcA expression in E. coli is higher under anaerobic than aerobic growth conditions, but the opposite is true in S. Typhimurium. Interestingly, a tdcR mutant strain of E. coli showed a similar expression pattern to that observed in S. Typhimurium and was also induced by anaerobic shock. These results suggest that the induction of tdcA expression by anaerobic conditions is observable when tdcA expression is low owing to the absence of TdcR.

• Journal of Industrial Technology
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• v.28 no.B
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• pp.59-63
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• 2008

Rapid production of therapeutic proteins such as angiostatin and endostatin angiogenic inhibititors has been highly demanded for cancer treatment. In this regard, recombinant human angiostatin and endostatin were successfully expressed as soluble forms by maltose binding protein (MBP)-mediated fusion expression in Escherichia coli. PCR amplified, angiostatin and endostatin genes from human placenta cDNA library were inserted into an expression vector pMAL-c2e to construct prokaryotic expression vectors, pMAL-c2e/AS and pMAL-c2e/ES, respectively. Recombinant angiostatin and endostatin were efficiently expressed in E. coli origami (DE3) after IPTG induction and protein expression were confirmed by SDS-PAGE analyses. The expressed recombinant proteins were purified near homogenity using an amylose affinty column chromatography. In contrast that previous E. coli expressions were all insoluble, our results first time demonstrated that MBP fused human angiostatin and endostatin were soluble in E. coli.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.550-557
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• 2010

Escherichia coli (E. coli) heat-labile enterotoxin B subunit (LTB) was regarded as one of the most powerful mucosal immunoadjuvants eliciting strong immunoresponse to coadministered antigens. In the research, the high-level secretory expression of functional LTB was achieved in P. pastoris through high-density fermentation in a 5-1 fermentor. Meanwhile, the protein was expressed in E. coli by the way of inclusion body, although the gene was cloned from E. coli. Some positive yeast and E. coli transformants were obtained respectively by a series of screenings and identifications. Fusion proteins LTB-6$\times$His could be secreted into the supernatant of the medium after the recombinant P. pastoris was induced by 0.5% (v/v) methanol at $30^{\circ}C$, whereas E. coli transformants expressed target protein in inclusion body after being induced by 1 mM IPTG at $37^{\circ}C$. The expression level increased dramatically to 250-300 mg/l supernatant of fermentation in the former and 80-100 mg/l in the latter. The LTB-6$\times$His were purified to 95% purity by affinity chromatography and characterized by SDS-PAGE and Western blot. Adjuvant activity of target protein was analyzed by binding ability with GMI gangliosides. The MW of LTB-6$\times$His expressed in P. pastoris was greater than that in E. coli, which was equal to the expected 11 kDa, possibly resulted from glycosylation by P. pastoris that would enhance the immunogenicity of co-administered antigens. These data demonstrated that P. pastoris producing heterologous LTB has significant advantages in higher expression level and in adjuvant activity compared with the homologous E. coli system.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1422-1428
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• 2006

An ABC transporter apparatus of the Gram-negative bacterial type I secretion pathway can be used as a secretory protein expression system in Escherichia coli. Four types of coexpression systems for the Pseudomonas fluorescens lipase gene, tliA, and its cognate ABC transporter gene cluster, tliDEF, were constructed. When the relative expression levels were changed by adding different concentrations of IPTG, the secretion (16.9 U/ml of culture) of TliA in E. coli [pTliDEFA-223+pACYC184] was significantly higher than E. coli [pKK223-3+pTliDEFA-184] secreting the lowest level of TliA (5.2 U/ml of culture). Maximal accumulation of the lipase secreted occurred in the mid-exponential phase, implying that the efficient protein secretion via an ABC transporter was restricted only to actively growing cells. Finally, the secretion level of TliA in E. coli [pTliDEFA-223+pACYC184] was increased to 26.4 U/ml by inducing gene expression at the culture initiation time. These results indicate that a significant increase in the ABC transporter-dependent protein secretion can be achieved by simply controlling the relative expression levels between the ABC transporter and its passenger protein, even in the recombinant E. coli cells.

• Korean Journal of Microbiology
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• v.29 no.2
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• pp.80-84
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• 1991

We cloned and expressed hepatitis B viral core antigen (HBcAg) gene in E. coli using $P_{L}$ promoter system. For optimal expression of the gene, we undertook the studies on the effects of the distance between Shine-Dalgarno (SD) sequence and start codon, copy number of repressor gene, induction temperature, and the stability of the core antigen. The results demonstrated that the induction at 37.deg.C was more efficient than at 42.deg.C, and the 11 base pairs (bp) distance between SD sequence and start codon of HBcAg gene was more efficient than the 15 bp distance in E. coli. The copy number of cI857 repressor gene did not influence on the expression of HBcAg, and the expression level of HBcAg in mutant type (low protease activity) and wild type strains was almost the same. The produced core antigen appeared to be HBcAg not HBeAg judged by two different radioimmunoassat (RIA) kits. This result suggested that the antigen was stable in E. coli.i.

• Journal of Microbiology and Biotechnology
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• v.25 no.2
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• pp.247-254
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• 2015

In this presentation, I describe the expression and purification of the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a commercially available double cistronic vector, pACYCDuet-1, to express the receptor heterodimer in a single cell as the soluble form. I describe here the expression and characterization of a biologically active heterodimer composed of the liver X receptor β-ligand binding domain and retinoid X receptor α-ligand binding domain. Although many of these proteins were previously seen to be produced in E. coli as insoluble aggregates or "inclusion bodies", I show here that as a form of heterodimer they can be made in soluble forms that are biologically active. This suggests that co-expression of the liver X receptor β-ligand binding domain with its binding partner improves the solubility of the complex and probably assists in their correct folding, thereby functioning as a type of molecular chaperone.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.750-753
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• 2001

Proteomics is a formalized approach for obtaining a rapid snap-shot of the protein complement of a tissue, cell or cell component. Such an approach is powerful in that it allows a parallel assessment of temporal protein fluxes. This is an important concept in view of the dynamic nature of protein expression. Undoubtedly, changes in protein expression are essential in any study aimed at investigating cellular networks. In this study, we analyzed and compared the proteomes of recombinant E. coli strain before and after induction. Proteome expression patterns of recombinant E. coli were resolved on 2D-gels, and the variations in the relative expression level of particular proteins were examined using software-aided protein quantification tool. We observed above 800 spots on a 2D-gel using Melanie II software. Many proteins which involved in chaperones were significantly up-regulated in recombinant E. coli. Therefore, it could be concluded that the expression of recombinant protein in E. coli acted as a stress to the cells, which change cells ability to synthesize proteins and induced the expression of various protective proteins.

• BMB Reports
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• v.33 no.2
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• pp.166-171
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• 2000

Thioredoxin is a multifunctional protein that is ubiquitous in microorganisms, animals and plants. Previously, the expression of the Escherichia coli thioredoxin gene (trxA) was found to be negatively regulated by cAMP. In the present study, the effect of temperature on the expression of the E. coli trxA gene was investigated. In order to examine the temperature effect, the fusion plasmid pCL70 that harbors the E. coli trxA P1P2 promoter was used. The other two fusion plasmids, pJH3 and pMH521 that were constructed in different vectors which harbor the E. coli trxA P2 promoter, were also used. When the E. coli strain MC1061/pCL70 was grown in a rich medium at $25^{\circ}C$, $34^{\circ}C$ and $42^{\circ}C$, the cells grown at $42^{\circ}C$ gave the highest $\beta$-galactosidase activity. The E. coli MC1061/pJH3 and MC1061/pMG521 cells showed increased $\beta$-galactosidase activity after the shift of the culture temperature to $42^{\circ}C$. The wild-type trxA gene of the E. coli MC1061 cells produced much higher thioredoxin activity at the higher temperature. These results support the conclusion that the E. coli trxA gene is regulated in a temperature-dependent manner. Especially the expression from its P2 promoter appeared to be sensitive to temperature.

• Journal of Life Science
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• v.11 no.2
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• pp.108-110
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• 2001

The E. coli expression system to overproduce a harmful protein, carboxypeptidase Taq was developed. Since expression plasmid pCK305N containing the colicin promoter already has the initiation codon on the restriction site, the initiation codon of the CPase Taq gene was removed. Expression plasmid pCP4-col includes the entire CPase Taq gene, which is directed by the colicin promoter. E. coli cells harboring pCP-col produced a high amount of the enzyme when they were cultured in the present of mitomycin C (0.4 ${\mu}g$/ml). An amount of purified enzyme produced by pCP4-col directed by the colicin promoter was 10.5 mg. This result indicated that the novel E. coli expression system controlled by the colicin promoter could produce almost twice amounts of CPase Taq than the conventional system controlled by the tart promoter.

• Archives of Pharmacal Research
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• v.21 no.3
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• pp.305-309
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• 1998

Human cytochrome P450 1A2 is one of the major cytochrome P450s in human liver. It is known to be capable of activating a number of carcinogens such as arylamines and heterocyclic amines. In order to develop the new bacterial mutagenicity test system with human P450, a full length of human P450 1A2 cDNA inserted into pCW bacterial expression vector was introduced to Escherichia coli WP2 uvrA strain which is a well-known E. coli strain for bacterial reverse mutagenicity assay. Expressed human P450 1A2 showed typical P450 hemoprotein spectra. Maximum expression was achieved at 48 hrs after incubating at $30^{\circ}C$ in terrific broth containing ampicillin, IPTG and other supplements. High level expression of P450 1A2 in E. coli WP2 uvrA membranes was determined in SDS-PAGE. The well-known mutagens 2-aminoanthracene and MElQ increased the revertant colonies of E. coli WP2 uvrA expressing human P450 1A2 without an exogenous rat hepatic post-mitochondrial supernatant (S9 fraction) in a dose-dependent manner. The results show that the functional expression of human P450 in bacterial mutagenicity tester strain will provide a useful tool for studying the mechanism of the mutagenesis and carcinogenesis of new drugs and environmental chemicals.