• Animal cells and systems
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• v.16 no.6
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• pp.455-461
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• 2012

Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase (GST) gene as a fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells using a pGEX-4T-1 expression vector, and the fusion protein was induced by ioporpyl-${\beta}$-D-thiogalactopyranoside (IPTG). The recombinant protein produced as an inclusion body was resolubilized in solubilization buffer, and the resultant solution was dialyzed in refolding buffer. After thrombin cleavage, the recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoreactivity against the anti-Agrius lysozyme was observed by western blot analysis of this protein. The recombinant lysozyme displayed antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was confirmed by the inhibition zone assay.

• Microbiology and Biotechnology Letters
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• v.20 no.6
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• pp.637-641
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• 1992

The promoter regions from chromosomal DNA of Bacillus sp. SSA3 which is responsible for fermentation of Korean traditional soy sauce, were cloned for construction of expression vector of Bacills sp. SSA3. Recombinant plasmids were constructed by insertion of HindIIl-cleaved Bacillus sp. SSA3 chromosomal DNA fragments in front of the CAT gene of pGR71 plasmid and B-galactosidase gene of pUC18 plasmid. 6 recombinant plasmids were isolated from chloramphenicol resistant E. coli JM109 clones. All these plasmids were found to have promoter activity in Bacills sp. SSA3 and E. coli JM109. When these 6 clones of Bacills sp. SSA3 were cultivated in LB agar medium supplemented with 10% NaCI. fused CAT gene expression of 4 clones was significantly decreased in common. But the others were poorly inhibited.

• Journal of Microbiology and Biotechnology
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• v.17 no.3
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• pp.474-480
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• 2007

Fusarium wilt of cucumbers was effectively controlled by Escherichia coli expressing an endochitinase gene (chiA), and the rate was as effective (60.0%) as the wild-type strain S. proteamaculans 3095 (55.0%) where the gene was cloned. However, live cells of soil inoculated E. coli host harboring the chiA gene did not proliferate but declined 100-fold from $10^8$ CFU during the first week and showed less than 10 cells after day 14, suggesting that E. coli was able to express and produce the chitinase enzyme to the soil even as the population was gradually decreasing. Because the majority of the strains was alive for only a short period of time and the Fusarium-affected seedlings showed symptoms of wilting within 7-10 days, it seems that the pathogen control was decided early after the introduction of the biocontrol agent, eliminating the survival of the antagonist. These results indicated that soil inoculated E. coli could sufficiently express and produce the recombinant protein to control the pathogen, and root or soil colonization of the antagonist might not be a significant factor in determining the efficacy of biological control.

• Journal of Microbiology and Biotechnology
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• v.19 no.7
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• pp.727-733
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• 2009

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-n-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite,N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other Histagged proteins expressed 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.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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• pp.275-280
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• 2000

The inulin fructotransferse (depolymerizing) (IFTase, EC 2.4.1.93) gene of Arthrobacter sp. A-6 was cloned and expressed in Escherichia coli and Bacillus subtilis. The IFTase gene consisted of an ORF of 1.311 nucleotides encoding a polypeptide of 436 amino acids containing a signal peptide of 31 amino acids in the N-terminus. The molecular mass of the IFTase based on the nucleotide sequence was calculated to be 46.116 Da. The recombinant E. coli $DH5{\alpha}$ cells expressing the Arthrobacter sp. A-6 IFTase gene produced most of the IFTase intracelularly. In contrast, the recombinant B. subtilis DB 104 carrying the IFTas gene on a B. subtilis-E. Coli expression vector secreted the IFTase into the culture fluid efficiently.

• BMB Reports
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• v.30 no.1
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• pp.60-65
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• 1997

Expression of human Cu.Zn-superoxide dismutase (SOD) with activity comparable to human erythrocyte enzyme was achieved in E. coli B21(DE3) by using the pET-17b expression vector containing a T7 promoter. Recombinant human SOD was found in the cytosol of disrupted bacterial cells and represented > 25% of the total bacterial proteins. The protein produced by the E. coli cells was purified using a combination of ammonium sulfate precipitation, Sephacryl S-100 gel filtration and DEAE-Sephacel ion exchange chromatography. The recombinant Cu,Zn-SOD and human erythrocyte enzyme were compared using dismutation activity, SDS-PAGE and immunoblotting analysis. The mass of the subunits was determined to be 15,809 by using a electrospray mass spectrometer. The copper specific chelator. diethyldithiocarbamate (DOC) reacted with the recombinant Cu,Zn-SOD. At $50{\mu}M$ and $100{\mu}M$ concentrations of DOC, the dismutation activity was not inhibited for one hour but gradually reduced after one hour. This result suggests that the reaction of DOC with the enzyme occurred in two distinct phases (phase I and phase II). During phase I of this reaction, one DOC reacted with the copper center, with retention of the dismutation activity while the second DOC displaced the copper, with a loss of activity in phase II.

• Archives of Pharmacal Research
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• v.15 no.1
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• pp.48-51
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• 1992

Achromobacter xylosoxidans KF701 and Pseudomonas putida (NAH7) were significantly different in degradative capability of aromatic compounds including benzoates, biphenyls, and naphthalene. However, both of the bacterial strains can grown on catechol as the sole carbon and energy source. Catechol 2, 3-dioxygenase gene for naphthalene oxidation or biphenyl oxidation was cloned into Escherichia coli HB 701. A E. coli HB 101 clone containing catechol 2, 3-dioxygenase gene from P. putida (NAH7) contains a recombinant plasmid with 3.60kb pBR322 and 6-kb insert DNA. Another E. coli HB101 clone containing catechol 2, 3-dioxygenase gene from A. xylosoxidans KF 701 has a recombinant plasmid with 4.4kb pBR322 and 10-kb insert DNA. Physical maps of the recombinant plasmids were constructed, and catechol 2, 3-dioxygenase gene in the recombinant plasmide was further localized and subcloned int M13. The cloned-catechol 2, 3-dioxygenase game products were identified as yellow bands on nondenaturaing polyacrylamide gel after electrophoresis followed by activity staining with catechol solution.

• BMB Reports
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• v.37 no.5
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• pp.556-564
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• 2004

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2% glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.

• KSBB Journal
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• v.26 no.2
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• pp.119-125
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• 2011

In the synthesis of nanoparticles, much attention has been paid to regulating the particle size. There has been a possible evident that using the central cavity (core) of the protein ferritin has a greatly significant influence on it because the core can generate the nanometer-sized mineral particles of variable metal ions. In this report, recombinant human L-ferritins produced from Saccharomyces cerevisiae were purified and their molecular properties were characterized. The cDNA for human ferritin L chain was also expressed in another host such as Escherichia coli, and the properties of recombinant L-ferritins were compared. From isoelectric focusing experiment, the L-ferritin from the recombinant yeast showed no indication of N-glycosylation. Some post-translational modifications other than N-glycosylation were speculated in the L-ferritins from yeast. A difference was made in the L-ferritins in their iron uptake rates and the initial rate of the L-ferritin from yeast was slightly increased. The reconstitution yield and size distribution of the core minerals were analyzed in the L-ferritins by transmission electron microscopy. The L-ferritin from yeast with higher reconstitution yield (54.5%) showed slightly larger sizes (mean 6.92 nm) with narrower size distribution than the L-ferritin from E. coli. It is, in conclusion, speculated that L-ferritin from yeast is relatively superior to the other, in view of the size of nanoparticle and its relative homogeneity.