• 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.

• Korean Journal of Microbiology
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• v.22 no.3
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• pp.167-173
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• 1984

The cellabiase (${\beta}$-glucosidase) gene in a Cellulomonas sp. CS1-1 was cloned into E. coli HB101 using the vector plasmid pBR322, and the expression of the gene in E. coli studied. The chromosomal DNA of the cellulomonas was digested by seveal restriction enzymes, each of which has only one cleaving site in plasmid pBR322. The recombinant plasmid, pSB2, created with Sal I frament, was expressed for the cellobiase gene in E. coli. The recombiant plasmid was estimated to contain 6.4 Kb foreign DNA at the Sal I site of plasmid pBR322 and the inserted DNA was mapped by single and double digestion with several enzymes. E. coli HB101(pSB2) has slowly grown in a mineral liquid medium containing cellobiose as a sole carbon source. The cellobiase activity in the transformed E. coli was 132 units per liter, which is equivalent to one twenty fifth of that in doner strain Cellulomonas sp. CS1-1. The transforned cell with plasmid containing cellulase gene grow well in the LB mediuns. The synthesis of cellobiase in the strain, E. coli HB101 (pSB2), was inhibited by glucose and at high concentration of cellobiose, and induced by cellobiose at low concentration.

• Journal of Microbiology and Biotechnology
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• v.25 no.6
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• pp.887-892
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• 2015

Uricase is an important microbial enzyme that can be used in the clinical treatment of gout, hyperuricemia, and tumor lysis syndrome. A total of 127 clinical isolates of Pseudomonas aeruginosa were tested for uricase production. A Pseudomonas strain named Ps43 showed the highest level of native uricase enzyme expression. The open reading frame of the uricase enzyme was amplified from Ps43 and cloned into the expression vector pRSET-B. Uricase was expressed using E. coli BL21 (DE3). The ORF was sequenced and assigned GenBank Accession No. KJ718888. The nucleotide sequence analysis was identical to the coding sequence of uricase gene puuDof P. aeruginosa PAO1. We report the successful expression of P. aeruginosa uricase in Escherichia coli. E. coli showed an induced protein with a molecular mass of about 58 kDa that was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. We also established efficient protein purification using the Ni-Sepharose column with activity of the purified enzyme of 2.16 IU and a 2-fold increase in the specific activity of the pure enzyme compared with the crude enzyme.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.92-96
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• 2001

Effect of environmental factors on the expression of soluble forms of Bacillus macerans cyclodextrin glycosyltransferase in recombinant Escherichia coli BL21(DE3)pLysE:pTCGT1 were investigated. The amount of soluble CGTase produced in the cell was measured by determining its enzymatic activity. The soluble fractionof the enzyme was increased by lowering the culture temperature to $30{\circ}C$ and medium pH to 5.8 compared to the enzyme production in LB medium at $37^{\circ}C$ and pH7.0. Addition of 0.2 M NaCl enhanced enzyme expression levels at the expense of cell growth. Glycine betaine that was added after 3 h of induction protected not only the cell growth from hig osmotic pressue but also hepld in vivo folding of CGTase in recombinant E. coli. Addition of 1 mM $CaCl_2$ was also effective in the expression of soluble CGTase, resulting in 15 U/ml of the enzyme activity.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.451-455
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• 1996

Physiological studies on the expression of Bacillus thuringiensis subsp. tenebrionis (Btt) gene coding for insecticidal protein in recombinant Escherichia coli 537 were carried out to identify optimal culture condition. It was necessary to shift culture temperature from 30 to $42^{\circ}C$ to express the gene. Expression of the Btt toxin gene by recombinant E. coli 537 began within one hour after induction. Complex nitrogen sources increased production of the insecticidal protein. The total insecticidal protein was 0.5 g/I when using yeast extract as a complex nitrogen source. Soybean hydrolysate showed apparently the highest induction efficiency. After induction, the cellular content of the insecticidal protein was 5.4 times higher than it had been before induction. The optimal cultivation strategy was found to grow cells for 7hours at $30^{\circ}C$ and then 5-8 hours at $42^{\circ}C$. The optimal cultivation pH for the production of insecticidal protein was 6.5. The Btt toxin produced by the recombinant E. coli 537 was found to have the same level of potency against Colorado potato beetle as the original toxin.

• Journal of Microbiology and Biotechnology
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• v.24 no.1
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• pp.70-79
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• 2014

In an attempt to develop a variety of expression vector systems for Corynebacterium glutamicum, six types of promoters, including $P_{tac}$, $P_{sod}$, $P_{sod}$ with a conserved Shine-Dalgarno (SD) sequence from C. glutamicum, $P_{ilvC}$, $P_{ilvC}$ with a conserved SD-1 ($P_{ilvC-M1}$), and $P_{ilvC}$ with a conserved SD-2 ($P_{ilvC-M2}$), were cloned into a modified shuttle vector, pCXM48. According to analysis of promoter strength by quantitative reverse transcription PCR, $P_{sod}$ and $P_{sod-M}$ were superior to tac and ilvC promoters in terms of transcription activity in C. glutamicum. All of the promoters have promoter activities in Escherichia coli, and $P_{sod-M}$ displayed the highest level of transcriptional activity. The protein expression in constructed vectors was evaluated by measuring the fluorescence of green fluorescent protein (GFP) and SDS-PAGE. C. glutamicum harboring plasmids showed GFP fluorescence with an order of activity of $P_{ilvC}$ > $P_{ilvC-M1}$ > $P_{sod}$ > $P_{ilvC-M2}$ > $P_{sod-M}$, whereas all plasmids except pCSP30 with $P_{sod}$ displayed fluorescence activities in E. coli. Of them, the strongest level of GFP was observed in E. coli with $P_{sod-M}$, and this seems to be due to the introduction of the conserved SD sequence in the translational initiation region. These results demonstrate that the expression vectors work well in both C. glutamicum and E. coli for the expression of target proteins. In addition, the vector systems harboring various promoters with different strengths, conserved SD sequences, and multiple cloning sites will provide a comfortable method for cloning and gene expression, and consequently contribute to the metabolic engineering of C. glutamicum.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.403-411
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• 2005

The relationship between plasmid (~9.5 kb) and pediocin PA-1 in P. acidilactici K10 was confirmed by plasmid curing. The pediocin operon of P. acidilactici K10 was amplified by PCR (polymerase chain reaction), and the nucleotide sequence was analyzed. The sequence of the pediocin operon of P. acidilactici K10 was similar to those of P. acidilactici strains producing pediocin PA-1/ AcH. For the expression of pediocin PA-1 in E. coli, a pQEPED (pQE-30 Xa::mature pedA) was constructed. His-tagged recombinant pediocin PA-1 (-6.5 kDa) was translated by cell-free in vitro transcription and translation using pQEPED as a DNA template. Theresult of slot blotting assay showed that transcription of recombinant pedA in E. coli M15 was induced by the addition of isopropyl-$\beta$-D-thiogalactopyranoside (IPTG) at the final concentration of 1 mM. Although the recombinant pediocin PA-1 inhibited the growth of E. coli, it was expressed in the host strain and purified by nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography under denaturing condition. This is the first report for the production and one-step purification of biologically active recombinant pediocin PA-1 in E. coli.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.12
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• pp.1751-1757
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• 2004

Recombinant heterologous proteins can be produced as insoluble aggregates partially or perfectly inactive in Escherichia coli. One of the strateges to improve the solubility of recombinant proteins is fusion with a partner that is excellent in producing soluble fusion proteins. To improve the production of soluble $\beta$-galactosidase, the gene of Thermus thermophilus KNOUC112 $\beta$-galactosidase (KNOUC112 $\beta$-gal) was fused with thioredoxin gene, and optimization of its expression in E. coli TOP10 was performed. KNOUC112 $\beta$-gal in pET-5b was isolated out, fused with thioredoxin gene in pThioHis C, and transformed to E. coli TOP10. The $\beta$-galactosidase fused with thioredoxin was produced in E. coli TOP10 as dimer and trimer. The productivity of fusion $\beta$ -galactosidase expressed via pThioHis C at 37$^{\circ}C$ was about 5 times higher than that of unfused $\beta$-galactosidase expressed via pET-5b at 37$^{\circ}C$. Inclusion body of $\beta$-galactosidase was formed highly, regardless of the induction by IPTG when KNOUC112 $\beta$ -gal was expressed via pET-5b at 37$^{\circ}C$. Fusion $\beta$ -galactosidase expressed at 37$^{\circ}C$ via pThioHis C without the induction by IPTG was soluble, but the induction by IPTG promoted the formation of inclusion body. Lowering the incubation temperature for the expression of fusion gene under 25$^{\circ}C$ prevented the formation of inclusion body, optimally at 25$^{\circ}C$. 0.07 mM of IPTG was sufficient for the soluble expression of fusion gene at 25$^{\circ}C$. The soluble production of Thermus thermophilus KNOUC112 $\beta$-galactosidase could be increased about 10 times by fusion with thioredoxin, and optimization of incubation temperature and IPTG concentration for induction.

• Journal of Life Science
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• v.14 no.1
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• pp.121-125
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• 2004

The synergistic effect of lowered incubation temperature and CroEL/ES expression on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) was studied in recombinant E. coli. pTCGTl and pGroll carrying the cgt and groEL/ES genes under the control of T7 promoter and pzt-I promoter, respectively, were co-introduced. Tetracycline (10 ng/ml) and IPTG (1 mM) were added at the early-exponential phase (2 hr) and mid-exponential phase (3 hr). Low temperature cultivation at $25^{\circ}C$ with groEL/ES expression improved the activity of CGTase by two fold, compared to $37^{\circ}C$ cultivation without chaperones. SDS-PACE analysis revealed that about 69% of CGTase in the total CGTase protein was found in the soluble fraction by overexpression of GroEL/ES and cultivation at$25^{\circ}C$, whereas 20% of CGTase was detected in the soluble fraction when E. coli was cultivated at $37^{\circ}C$ without chaperone. The amount of soluble CGTase from $25^{\circ}C$ culture with chaperone was 3.5-fold higher than that of $37^{\circ}C$ culture without chaperone. Therefore the expression of CroEL/ES and low temperature cultivation greatly enhanced the soluble production of CGTase in E. coli.

• BMB Reports
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• v.36 no.5
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• pp.493-498
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• 2003

The scFv antibody towards the Burkholderia pseudomallei exotoxin was previously constructed by phage display and exhibited good specificity towards the exotoxin. We report here the optimization of the scFv expression in an E. coli expression system. Four different E. coli strains (ER2537, TG1, HB2151, and XL1-Blue) were examined for optimal expression of the scFv protein. Two types of carbon source (i.e. 0.2% glucose and 0.2% glycerol) were also tested for their ability to induce the scFv expression. Cells that carried the scFv construct were grown at $30^{\circ}C$ and induced with 0.05 mM IPTG. The expression was then monitored by SDS-PAGE, Western blotting, and indirect ELISA. The Western blot profile showed different levels of the scFv expression among the host strains; XL1-Blue exhibited the highest level of the scFv protein expression. Glycerol at a concentration of 0.2% (v/v) significantly increased the scFv protein expression level when compared to 0.2% (w/v) glucose. Further optimization demonstrated that the scFv protein expression in XL1-Blue was the most optimal with a glycerol concentration as low as 0.05%. However, by indirect ELISA, only the scFv protein that was expressed in 0.2% (v/v) glycerol exhibited high specificity towards the Burkholderia pseudomallei exotoxin.