• Microbiology and Biotechnology Letters
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• v.51 no.1
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• pp.37-42
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• 2023

Neutral and non-essential amino acid, glutamine (Gln), plays an essential role in supplying nitrogen to all the amino acids and nucleotides in the mammalian body. Gln is also the most important carbon source that provides intermediates for gluconeogenesis and fatty acid synthesis and supplements the tricarboxylic acid cycle in fast-growing cancer cells. Among the known 14 Gln transporter genes, soluted carrier family 1 member 5 (SLC1A5) has been reported to be closely associated with cancer cell growth. Three variants (v1, v2, and v3) have been derived from SLC1A5. Here, we established a heterologous gene expression system for the active form of human SLC1A5 variant-2 (hSLC1A5v2) in Escherichia coli. v2 is the smallest variant that has not yet been studied. Four expression systems were investigated: pBAD, pCold, pET, and pQE. We also addressed the problem of codon usage bias. Although pCold and pET overexpressed hSLC1A5v2 in E. coli, they were functionally inactive. hSLC1A5v2 using the pBAD system was able to catalyze the successful transport of Gln, even if it was not highly expressed. Initial activity of hSLC1A5v2 for [¹⁴C] Gln uptake in E. coli reached up to 6.73 μmole·min^-1·gDW^-1 when the cell was induced with 80 mM L-arabinose. In this study, we demonstrated a heterologous expression system for the human membrane protein, SLC1A5, in E. coli. Our results can be used for the functional comparison of SLC1A5 variants (v1, v2, and v3) in future studies, to facilitae the developement of SLC1A5 inhibitors as effective anticancer drugs.

• Journal of Microbiology and Biotechnology
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• v.17 no.2
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• pp.244-252
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• 2007

Escherichia coli has a PhoR-PhoB two-component regulatory system to detect and respond to the changes of environmental phosphate concentration. For the E. coli W3110 strain growing under phosphate-limiting condition, the changes of global gene expression levels were investigated by using DNA microarray analysis. The expression levels of some genes that are involved in phosphate metabolism were increased as phosphate became limited, whereas those of the genes involved in ribosomal protein or amino acid metabolism were decreased, owing to the stationary phase response. The upregulated genes could be divided into temporarily and permanently inducible genes by phosphate starvation. At the peak point showing the highest expression levels of the phoB and phoR genes under phosphate-limiting condition, the phoB- and/or phoR-dependent regulatory mechanisms were investigated in detail by comparing the gene expression levels among the wild-type and phoB and/or phoR mutant strains. Overall, the phoB mutation was epistatic over the phoR mutation. It was found that PhoBR and PhoB were responsible for the upregulation of the phosphonate or glycerol phosphate metabolism and high-affinity phosphate transport system, respectively. These results show the complex regulation by the PhoR-PhoB two-component regulatory system in E. coli.

• Animal cells and systems
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• v.15 no.1
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• pp.29-36
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• 2011

Insect lysozymes are basic, cationic proteins synthesized in fat body and hemocytes in response to bacterial infections and depolymerize the bacterial cell wall. The c-type lysozyme of the insect Spodoptera litura (SLLyz) is a single polypeptide chain of 121 residues with four disulfide bridges and 17 rare codons and is approximately 15 kDa. The full-length SLLyz cDNA is 1039 bp long with a poly(A) tail, and contains an open reading frame of 426 bp long (including the termination codon), flanked by a 54 bp long 5' UTR and a 559 bp long 3' UTR. As a host for the production of high-level recombinant proteins, E. coli is used most commonly because of its low cost and short generation time. However, the soluble expression of heterologous proteins in E. coli is not trivial, especially for disulfide-bonded proteins. In order to prevent inclusion body formation, GST was selected as a fusion partner to enhance the solubility of recombinant protein, and fused to the amplified products encoding mature SLLyz. The expression vector pGEX-4T-1/rSLLyz was then transformed into E. coli BL21(DE3)pLysS for soluble expression of rSLLyz, and the soluble fusion protein was purified successfully. Inhibition zone assay demonstrated that rSLLyz showed antibacterial activity against B. megaterium. These results demonstrate that the GST fusion expression system in E. coli described in this study is efficient and inexpensive in producing a disulfide-bonded rSLLyz in soluble, active form, and suggest that the insect lysozyme is an interesting system for future structural and functional studies.

• Biotechnology and Bioprocess Engineering:BBE
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• v.4 no.1
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• pp.63-65
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• 1999

Although many pharmaceutically useful proteins are produced in E. coli expression system, it is very are rare for the system to be used in the production of diagnostic antigen due to a major problem, i.e., false-positive reaction of e. coli host-derived proteins contaminating purified diagnostic antigen with human sera. The N (nucleocapsid) protein of Seoul virus causing haemorrhagic fever with renal syndrome (HFRS) was produced in E. coli BL21 (DE3), and used for the detection of N protein-specific antibodies in human sera. Using the N protein as a diagnostic antigen of HFRS, the false-positive reaction was cleared by merely mixing the test sera with the extract of E. coli host strain not harboring expression plasmid.

• International Journal of Industrial Entomology and Biomaterials
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• v.8 no.2
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• pp.145-149
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• 2004

The expression of a fusion protein comprised of the B. thuringiensis crystal protein, Cry1Ac, and enhanced green fluorescent protein (EGFP) in Escherichia coli XLl-blue was examined. Three recombinant plasmids were transformed into E. coli XL1-blue and named as ProAc/Ec, MuEGFP/Ec and ProMu-EGFP/Ec, respectively. All transformants were observed by light and fluorescence microscopy at mid-log phase. The expression in E. coli transformants, ProMu-EGFP/Ec and MuEGFP/Ec, exhibited bright enough fluorescence to be observed. Furthermore, ProMu-EGFP/Ec produced fluorescent inclusions, which may have been recombinant crystals between EGFP and Cry1Ac while MuEGFP/Ec expressed soluble EGFP in cell. In SDS-PAGE, ProAc/Ec had 130 kDa crystal protein band and MuEGFP/Ec had thick 27 kDa EGFP band. However, ProMu-EGFP/Ec had about 150 kDa fusion protein band. Accordingly, these results indicated that a fusion protein between the B. thuringiensis crystal protein and a foreign protein under the lacZ promoter was successfully expressed as granular structure in E. coli. It is suggested that the E. coli expression system by N-terminal fusion of B. thuringiensis crystal protein may be useful as excellent means for fusion expression and characterization of B. thuringiensis fusion crystal protein.

• Korean Chemical Engineering Research
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• v.58 no.2
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• pp.280-285
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• 2020

We have developed a constitutive display system of the Pseudomonas fluorescens SIK W1 TliA lipase on the cell surface of Escherichia coli using E. coli outer membrane protein C (OmpC) as an anchoring motif, which is an economical compared to induced system. For the constitutive expression of truncated OmpC-TliA fusion proteins, gntT104 promoter was employed. Cell growth was not affected by over expression of fusion protein during entire culture time, suggesting cell lysis was not a problem. The localization of truncated OmpC-TliA fusion protein on the cell surface was confirmed by immunofluorescence microscopy and measuring whole cell lipase activity. Constitutively displayed lipase was very stable, retaining activity enantioselectivity throughout the five repeated reactions. These results suggest that OmpC from E. coli be a useful anchoring motif for displaying enzymes on the cell surface without any inducers, and this stable surface display system can be employed for a broad range of biotechnological applications.

• KSBB Journal
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• v.23 no.5
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• pp.403-407
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• 2008

Recently, Candida antarctica lipase B (CalB) draws attention from industries for various applications for food, detergent, fine chemical, and biodiesel, because of its characteristics as an efficient biocatalyst. Since many industrial processes carry out in organic solvent and at high temperature, CalB, which is stable under harsh condition, is in demand from many industries. In order to reform CalB promptly, the expression system which has advantages of ease to use and low cost for gene libraries screening was developed using E. coli. The E. coli strains, Rosettagami with competence for enhanced disulfide bond formation, Novablue, and $DH5{\alpha}$, were exploited in this study. To obtain the soluble CalB, the pCold I vector expressing the cloned gene at $15^{\circ}C$ and the chaperone plasmids containing groES/groEL, groES/groEL/tig, tig, dnaK/dnaJ/grpE, and dnaK/dnaJ/grpE/groES/groEL were used for coexpression of CalB and chaperones. The colonies expressing functional lipase were selected by employing the halo plate containing 1% tributyrin, and the CalB expression was confirmed by SDS-PAGE. E. coli Rosettagami and $DH5{\alpha}$ harbouring groES/groEL chaperones were able to express soluble CalB effectively. From a facilitative point of view, E. coli $DH5{\alpha}$ is more suitable for further mutation study.

• 한국유가공학회:학술대회논문집
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• 2007.09a
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• pp.13-20
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• 2007

Bovine lactoferricin(LFcin B) is a peptide of 25 amino acids that originated from the N terminus of bovine lactoferrin, and is characterized as having potent antimicrobial activity against bacteria, fungi, protozoa and viruses. But, direct expression of Lfcin B is lethal to Escherichia coli. For the efficient production of Lfcin B in E. coli, we developed an expression system in which the gene for cationic Lfcin B was fused to an anionic peptide gene, and successfully expressed the concatemeric fusion gene in E. coli. The purified recombinant Lfcin B was found to have antimicrobial activity, as the native Lfcin B peptide does.

• 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.

• Journal of Microbiology and Biotechnology
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• v.32 no.8
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• pp.1047-1053
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• 2022

When ptsG, a glucose-specific phosphotransferase system (PTS) component, is deleted in Escherichia coli, growth can be severely poor because of the lack of efficient glucose transport. We discovered a new PTS transport system that could transport glucose through the growth-coupled experimental evolution of ptsG-deficient E. coli C strain under anaerobic conditions. Genome sequencing revealed mutations in agaR, which encodes a repressor of N-acetylgalactosamine (Aga) PTS expression in evolved progeny strains. RT-qPCR analysis showed that the expression of Aga PTS gene increased because of the loss-of-function of agaR. We confirmed the efficient Aga PTS-mediated glucose uptake by genetic complementation and anaerobic fermentation. We discussed the discovery of new glucose transporter in terms of different genetic backgrounds of E. coli strains, and the relationship between the pattern of mixed-acids fermentation and glucose transport rate.