• Journal of Microbiology and Biotechnology
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• v.21 no.6
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• pp.637-645
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• 2011

Production of recombinant proteins by excretory expression has many advantages over intracellular expression in Escherichia coli. Hyperexpression of a secretory exoglucanase, Exg, of Cellulomonas fimi was previously shown to saturate the SecYEG pathway and result in dramatic cell death of E. coli. In this study, we demonstrated that overexpression of the PspA in the JM101(pM1VegGcexL-pspA) strain enhanced excretion of Exg to 1.65 U/ml using shake-flask cultivation, which was 80% higher than the highest yield previously obtained from the optimized JM101(pM1VegGcexL) strain. A much higher excreted Exg activity of 4.5 U/ml was further achieved with high cell density cultivation using rich media. Furthermore, we showed that the PspA overexpression strain enjoyed an elevated critical value (CV), which was defined as the largest quotient between the intracellular unprocessed precursor and its secreted mature counterpart that was still tolerable by the host cells prior to the onset of cell death, improving from the previously determined CV of 20/80 to the currently achieved CV of 45/55 for Exg. The results suggested that the PspA overexpression strain might tolerate a higher level of precursor Exg making use of the SecYEG pathway for secretion. The reduced lethal effect might be attributable to the overexpressed PspA, which was postulated to be able to reduce membrane depolarization and damage. Our findings introduce a novel strategy of the combined application of metabolic engineering and construct optimization to the attainment of the best possible E. coli producers for secretory/excretory production of recombinant proteins, using Exg as the model protein.

• Journal of Microbiology
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• v.42 no.2
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• pp.111-116
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• 2004

It is known that Bacillus subtilis glutamyl-tRNA synthetase (GluRS) mischarges E. coli $tRNA_{1}$$^{Gln}$ with glutamate in vitro. It has also been established that the expression of B. subtilis GluRS in Escherichia coli results in the death of the host cell. To ascertain whether E. coli growth inhibition caused by B. subtilis GluRS synthesis is a consequence of Glu-$tRNA_{1}$$^{Gln}$ formation, we constructed an in vivo test system, in which B. subtilis GluRS gene expression is controlled by IPTG. Such a system permits the investigation of factors affecting E. coli growth. Expression of E. coli glutaminyl-tRNA synthetase (GlnRS) also amelio-rated growth inhibition, presumably by competitively preventing $tRNA_{1}$$^{Gln}$ misacylation. However, when amounts of up to 10 mM L-glutamine, the cognate amino acid for acylation of $tRNA_{1}$$^{Gln}$, were added to the growth medium, cell growth was unaffected. Overexpression of the B. subtilis gatCAB gene encoding Glu-$tRNA^{Gln}$ amidotransferase (Glu-AdT) rescued cells from toxic effects caused by the formation of the mis-charging GluRS. This result indicates that B. subtilis Glu-AdT recognizes the mischarged E. coli Glu-$tRNA_{1}$$^{Gln}$, and converts it to the cognate Gln-$tRNA_{1}$$^{Gln}$ species. B. subtilis GluRS-dependent Glu-$tRNA_{1}$$^{Gln}$ formation may cause growth inhibition in the transformed E. coli strain, possibly due to abnormal protein synthesis.

• Journal of Microbiology
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• v.34 no.2
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• pp.151-157
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• 1996

By PCR amplification using the sequence of the previously cloned shiga-like toxin II DNA, a gene encoding it has been cloned from an isolate of healthy Korean native bovine feces, Escherichia coli KSC109. The nucleotide sequence s included tow open reading frames coding for 319 and 89 amino acids corresponding to A and B subunits, respectively. Comparison of the nucleotide and predicted amino acid sequences of newly cloned gene (slt-II) with those of others in the SLT-II family revealed completely identical homology with SLT-II cloned previously from bacteriophabe DNA of E. coli 933 derived from a patient with hemorrhagic colities. In addition, the sequence homology of SLT-II with SLT-II variant form bovine was more than 95% at both the nucleotide and protein levels. Overexpression of SLT-II recombinant gene by induction with IPTG using an E, coli hostvector, system was conducted and the correctly processed products with active mature form exhibited 1000-fold higher cytotoxycity for Vero cells than that form original strain.

• Journal of Microbiology and Biotechnology
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• v.24 no.7
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• pp.969-978
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• 2014

The aprE2 gene with its prosequence from Bacillus subtilis CH3-5 was overexpressed in Escherichia coli BL21(DE3) by using plasmid pET26b(+). After IPTG induction, active and mature AprE2 was produced when cells were grown at $20^{\circ}C$, whereas inactive and insoluble enzyme was produced in a large amount when cells were grown at $37^{\circ}C$. The insoluble fraction was resuspended with 6 M guanidine-HCl and dialyzed against 2 M Tris-HCl (pH 7.0) or 0.5 M sodium acetate (pH 7.0) buffer. Then active AprE2 was regenerated and purified by a Ni-NTA column. Purified AprE2 from the soluble fraction had a specific activity of $1,069.4{\pm}42.4U/mg$ protein, higher than that from the renatured insoluble fraction. However, more active AprE2 was obtained by renaturation of the insoluble fraction. AprE2 was most stable at pH 7 and $40^{\circ}C$, respectively. The fibrinolytic activity of AprE2 was inhibited by PMSF, but not by EDTA and metal ions. AprE2 degraded $A{\alpha}$ and $B{\beta}$ chains of fibrinogen quickly, but not the ${\gamma}$-chain. AprE2 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-pNA. The $K_m$ and $k_{cat}/K_m$ of AprE2 was 0.56 mM and $3.10{\times}10^4S^{-1}M^{-1}$, respectively.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.598-598
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• 2003

• Applied Biological Chemistry
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• v.45 no.4
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• pp.190-194
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• 2002

Expression of Bacillus subtilis glutamyl-tRNA synthetase (GluRS) in Escherichia coli is lethal for the host, probably because this enzyme misaminoacylates ${tRNA_l}^{Gln}$ with glutamate in vivo. In order to overexpress B. subtilis GluRS, encoded by the gltX gene, in E. coli, this gene was amplified from B. subtilis 168 chromosomal DNA using PCR method and the entire coding region was cloned into a pET11a expression vector so that it was expressed under the control or the T7 Promoter. The resulting recombinant pEBER plasmid was transformed into E. coli Novablue (DE3) bearing the T7 RNA polymerase gene for expression. After IPTG treatment, the overproduced enzyme was purified using ammonium sulfate fractionation, Source Q anion exchange chromatography, Superdex-200 gel filtration, and Mono Q anion exchange chromatography. The purified enzyme yielded 18-fold increase in specific activity over the crude cell extract and its molecular weight was approximately 55 kDa on SDS-PAGE.

• Korean Journal of Microbiology
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• v.46 no.2
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• pp.223-227
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• 2010

RNase E plays a major role in the degradation and processing of a large number of RNA transcripts in Escherichia coli and forms the core component of the degradosome, a large protein complex involved in RNA metabolism. RraA and RraB are recently discovered protein inhibitors of RNase E and are evolutionarily conserved. In this study, we observed that, unlike RraA, overexpression of RraB did not rescue growth arrest of E. coli cells overexpressing RNase E. To examine whether this phenomenon stems from differential inhibitory effects of RraA and RraB on RNase E substrates, we analyzed three in vivo RNase E substrates. The results showed that RraA inhibited RNase E activity more efficiently than RraB on the degradation of RNA I, which controls the copy number of ColE1-type plasmid, and rpsO mRNA encoding ribosomal protein S15, while RraB was unable to inhibit the processing of pM1 RNA, a precursor of the RNA component of RNase P, by RNase E. Our results imply that RraB inhibits RNase E activity in a more substrate-dependent manner than RraA and this property of RraB may explain why overexpression of RraB could not rescue cells overexpressing RNase E from growth arrest.

• 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.

• Journal of Microbiology and Biotechnology
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• v.29 no.6
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• pp.944-951
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• 2019

Lipases are industrial enzymes that catalyze both triglyceride hydrolysis and ester synthesis. The overexpression of lipase genes is considered one of the best approaches to increase the enzymatic production for industrial applications. Subfamily I.2. lipases require a chaperone or foldase in order to become a fully-activated enzyme. The goal of this research was to isolate, clone, and co-express genes that encode lipase and foldase from Burkholderia territorii GP3, a lipolytic bacterial isolate obtained from Mount Papandayan soil via growth on Soil Extract Rhodamine Agar. Genes that encode for lipase (lipBT) and foldase (lifBT) were successfully cloned from this isolate and co-expressed in the E. coli BL21 background. The highest expression was shown in E. coli BL21 (DE3) pLysS, using pET15b expression vector. LipBT was particulary unique as it showed highest activity with optimum temperature of $80^{\circ}C$ at pH 11.0. The optimum substrate for enzyme activity was $C_{10}$, which is highly stable in methanol solvent. The enzyme was strongly activated by $Ca^{2+}$, $Mg^{2+}$, and strongly inhibited by $Fe^{2+}$ and $Zn^{2+}$. In addition, the enzyme was stable and compatible in non-ionic surfactant, and was strongly incompatible in ionic surfactant.

• Journal of Applied Biological Chemistry
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• v.43 no.4
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• pp.240-245
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• 2000

Two genes encoding maltooligosyltrehalose synthase (SaMTS) and maltooligosyltrehalose trehalohydrolase (SaMTH) were isolated from a hyperthermophilic microorganism, Sulfolobus acidocaldarius (ATCC 49462). ORFs of the SaMTS and SaMTH genes are 2,163 and 1,671 bp long and encode 720 and 556 amino acid residues, respectively. A bifunctional fusion enzyme (SaMTSH) was constructed through the gene fusion of SaMTS and SaMTH. Recombinant SaMTS, SaMTH, and SaMTSH fusion enzyme were overexpressed in E. coli BL21. SaMTS and SaMTH produced trehalose and maltotriose from maltopentaose in a sequential reaction. SaMTSH fusion enzyme catalyzed the sequential reaction in which the formation of maltotriosyltrehalose was followed by hydrolysis leading to the synthesis of trehalose and maltotriose. The SaMTSH fusion enzyme showed the highest activity at pH 5.0-5.5 and $70-75^{\circ}C$. SaMTS, SaMTH, and SaMTSH fusion enzyme were active in soluble starch, which resulted in the production of trehalose.