• BMB Reports
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• v.34 no.6
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• pp.502-508
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• 2001

A cDNA, encoding the human growth hormone (hGH), was synthesized based on the known 191 amino acid sequence. Its codon usage was optimized for a high level expression in Escherichia coli. Unique restriction sites were incorporated throughout the gene to facilitate mutagenesis in further studies. To minimize an initiation translation problem, a 624-bp cassette that contained a ribosome binding site and a start codon were fused to the hGH-coding sequence that was flanked between the EcoRI and HindIII sites. The whole fragment was synthesized by an overlapped extension of eight long synthetic oligonucleotides. The four-short duplexes of DNA, which were first formed by annealing and filling-in with a Klenow fragment, were assembled to form a complete hGH gene. The hGH was cloned and expressed successfully using a pET17b plasmid that contained the T7 promoter. Recombinant hGH yielded as much as 20% of the total cellular proteins. However, the majority of the protein was in the form of insoluble inclusion bodies. N-terminal amino acid sequencing also showed that the hGH produced in E. coli contained formyl-methionine. This study provides a useful model for synthesis of the gene of interest and production of recombinant proteins in E. coli.

• Journal of Microbiology and Biotechnology
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• v.30 no.3
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• pp.333-340
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• 2020

Macrophages are the cells of the first-line defense system, which protect the body from foreign invaders such as bacteria. However, Gram-negative bacteria have always been the major challenge for macrophages due to the presence of lipopolysaccharides on their outer cell membrane. In the present study, we evaluated the effect of phloretin, a flavonoid commonly found in apple, on the protection of macrophages from Escherichia coli infection. RAW 264.7 cells infected with standard E. coli, or virulent E. coli K1 strain were treated with phloretin in a dose-dependent manner to examine its efficacy in protection of macrophages. Our results revealed that phloretin treatment reduced the production of nitric oxide (NO) and generation of reactive oxygen species along with reducing the secretion of proinflammatory cytokines induced by the E. coli and E. coli K1 strains in a concentration-dependent manner. Additionally, treatment of phloretin downregulated the expression of E. coli-induced major inflammatory markers i.e. cyclooxygenase-2 (COX-2) and hemeoxygenase-1 (HO-1), in a concentration dependent manner. Moreover, the TLR4-mediated NF-κB pathway was activated in E. coli-infected macrophages but was potentially downregulated by phloretin at the transcriptional and translational levels. Collectively, our data suggest that phloretin treatment protects macrophages from infection of virulent E. coli K1 strain by downregulating the TLR4-mediated signaling pathway and inhibiting NO and cytokine production, eventually protecting macrophages from E. coli-induced inflammation.

• Journal of Microbiology
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• v.44 no.3
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• pp.293-300
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• 2006

The Escherichia coli heat-labile enterotoxin B subunit (HLT-B) is one of the most powerful mucosal immunogens and known mucosal adjuvants. However, the induction of high levels of HLT-B expression in E. coli has proven a difficult proposition. Therefore, in this study, the HLT-B gene was cloned from pathogenic E. coli and expressed as a fusion protein with GST (glutathion S-transferase) in E. coli BL2l (DE3), in an attempt to harvest a large quantity of soluble HLT-B. The culture conditions, including the culture media used, temperature, pH and the presence of lactose as an inducer, were all optimized in order to obtain an increase in the expression of soluble GST-rHLT-B. The biological activity of the purified rHLT-B was assayed in a series of GMI-ELISA experiments. The findings of these trials indicated that the yield of soluble recombinant GST-rHLT-B could be increased by up to 3-fold, as compared with that seen prior to the optimization, and that lactose was a more efficient alternative inducer than IPTG. The production of rHLT-B, at 92 % purity, reached an optimal level of 96 mg/l in a 3.7 L fermentor. The specific GM1 binding ability of the purified rHLT-B was determined to be almost identical to that of standard CTB.

• Journal of Food Hygiene and Safety
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• v.6 no.3
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• pp.133-137
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• 1991

In order to increase the expression of XMP aminase [EC 6.3.4.1], which catalizes the conversion of 5'-XMP to the DNA fragment containing gua A gene coding for XMP aminase from pLC 34-10 plasmid was subcloned into pBR 322, and 1.7 kb gua A gene fragment was recloned under the control of trp promoter of pDR 720, E. coli expression vector. XMP aminase activity had increased by about 17 times when compared with that of the strain earring pLC 34-10.

• Microbiology and Biotechnology Letters
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• v.40 no.2
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• pp.92-97
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• 2012

Antimicrobial peptides (AMPs) are important components of living organisms acting against Gram-negative and Gram-positive bacterial and fungal pathogens. Cathelicidin human peptides have a variety of biological activities that can be used in clinical applications. AMPs are not produced naturally in large quantities, and chemical synthesis is also economically impractical, especially for long peptides. Therefore, as an alternative, heterologous expression of AMPs by recombinant techniques has been studied as a means to reduce production costs. E. coli is an excellent host for the expression of AMPs, as well as other recombinant proteins, because of the low cost involved and its easy manipulation. However, overexpression of AMPs in E. coli has been shown to cause difficulties resulting from the toxicity of the subsequently produced AMPs. Therefore, fusion expression was theorized to be a solution to this problem. In this study, AMPs were expressed as fused proteins with the glutathione S-transferase (GST) binding protein to protect against the toxicity of AMPs when expressed in E. coli. The LL37, and hybrid gaegurin and LL37 (GGN4(1-16)-LL37(17-32), which we designated as GL32, peptides were expressed as GST-fusion proteins in E. coli and the fusion proteins were then purified by affinity columns. The purified peptides were obtained by removal of GST and were confirmed by western blot analysis. The purified antimicrobial peptides then demonstrated antimicrobial activities against Gram-negative and Gram-positive bacterial strains.

• Proceedings of the Korean Society of Toxicology Conference
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• 2001.05a
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• pp.143-143
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• 2001

Human cytochrome P450 (CYP or P450) 3A4 (CYP3A4) is the most abundant among P450s in human liver. We previously reported that the expression of CYP3A4 in membranes prepared from E. coli coexpressed the bicistronic construct of CYP3A4 and NADPH-P450 reductase with cytochrome b$_{*}$ (b5) was showed 20-60% higher than that in membranes from E. coli expressed only the bicistronic construct with culturing longer times (48-72h).(omitted)

• Korean Journal of Microbiology
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• v.27 no.4
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• pp.317-322
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• 1989

A gene can be selectively overexpressed in E. coli by utilizing the phage T7 RNA polymerase's stringent recognition and active transcription of the T7 promoter. The T7 expression system was constructed such that the T7 RNA polymerase gene is under the control of lacUV5 promoter in one plasmid, and that the target gene, the promoterless chloramphenicol acetyltransferase (CAT) gene with E. coli ribosome binding site is under the control of T7 promoter in the other plasmid. Only the E. coli cells containing both plasmids show high resistance to chloramphenicol. When the copy number of the runaway plasmid containing the polymerase gene was varied by a temperature shift, amounts of the CAT protein synthesized upon induction was correspondingly changed as shown in SDS gel electrophoresis.

• Journal of Microbiology and Biotechnology
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• v.13 no.4
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• pp.552-559
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• 2003

The FimH subunit of type 1-fimbriated Escherichiu coli (E. coli) has been determined as a major cause for urinary tract infections. Thus, to produce a possible vaccine antigen against urinary tract infections, the fimIH gene was genetically coupled to the ctxa2b gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimH/ctxa2b was then transformed into E. coli K-12 TB1 and its nucleotide sequence was verified. A fusion protein, based on fusing adhesin to the cholera toxin subunit A2B (CTXA2B), was induced with 0.01 mM isopropyl-${\beta}-D-thiogalactoside$ (IPTG) for 4 h at $37^{\circ}C$ to yield a soluble fusion protein. The fusion protein was then purified by affinity chromatography. The expressed fusion protein was confirmed by SDS-PAGE and Western blotting using antibodies to the maltose binding protein (MBP) or the cholera toxin subunit B (CTXB), plus the N-terminal amino acid sequence was also analyzed. The orderly-assembled fusion protein was confirmed by a modified $G_{Ml}-ganglioside$ ELISA, using antibodies to adhesin. The results indicated that the purified fusion protein was an adhesin/CTXA2B protein containing E. coli adhesin and the $G_{Ml}-ganglioside$ binding activity of CTXB. Accordingly, this adhesin/CTXA2B protein may be a potential antigen for oral immunization against uropathogenic E. coli.

• Journal of Microbiology and Biotechnology
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• v.23 no.3
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• pp.322-328
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• 2013

In this study, we show the expression and purification of the human recombinant farnesoid X receptor (FXR)- ligand binding domain (LBD) protein in E. coli using a double cistronic vector, pACYCDuet-1, as a soluble form. We describe here the expression and characterization of a biologically active $FXR-LBD_{(248-476)}$. When expressed in the influence of bacterial promoters ($P_{T7}$ and $P_{Tac}$) of the single cistronic expression vectors, the human recombinant $FXR-LBD_{(248-476)}$ was found to be totally insoluble. However, by using a double cistronic expression vector, we were able to obtain the human recombinant $FXR-LBD_{(248-476)}$ in a soluble form. To allow for biological activities, we have subcloned into the pACYCDuet-1 vector, expressed in E. coli cells at some optimized conditions, and purified and characterized the human recombinant active $FXR-LBD_{(248-476)}$ proteins using the fluorescence polarization assay. This suggests that the expression of FXR-LBD in a double cistronic vector improves its solubility and probably assists its correct folding for the biologically active form of the proteins. We suggest that this may represent a new approach to high expression of other nuclear receptors and may be useful as well for other classes of heterodimeric protein partners.

• Food Science and Biotechnology
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• v.17 no.3
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• pp.655-658
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• 2008

Although arabinose isomerase (E.C. 5.3.1.4), a commercial enzyme for edible tagatose bioconversion, can be expressed in an Escherichia coli system, this expression system might leave noxious by-products in food. To develop an eligible tagatose bioconversion with food-safe system, we compared the tagatose production activity of immobilized arabinose isomerase expressed in Bacillus subtilis (a host generally recognized as safe) with that of the enzyme expressed in E. coli. A 48% increase in tagatose production (4.3 g tagatose/L at $69.4\;mg/L{\cdot}hr$) was found using the B. subtilis-expressed immobilized enzyme system, compared to the E. coli-expressed enzyme system (2.9 g tagatose/L). The increased productivity with safety of the B. subtilis-expressed arabinose isomerase suggests that it is a more eligible candidate for commercial tagatose production.