• Proceedings of the PSK Conference
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• 2003.10b
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• pp.160.1-160.1
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• 2003

In order to confirm the biological function of ORF10 in cephabacin biosynthesis gene cluster of Lysobacter lactamgenus as an ATP-binding-cassette (ABC) transporter, the gene for ORF10 was amplified and subcloned into pET-28a(+) expression vector. After gene induction with 0.5 mM IPTG at 30~! and further cultivation at $30^~$ !. for 8 hr, a lot of the recombinant ORF10 protein was produced as soluble form in cytoplasmic fraction as well as a membrane protein in the membrane fraction as likely as other ABC transporters. (omitted)

• BMB Reports
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• v.40 no.4
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• pp.511-516
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• 2007

A glutathione S-transferase (GST) related to the phi (F) class of enzymes only found in plants has been cloned from the Oryza sativa. The GST cDNA was cloned by PCR using oligonucleotide primers based on the OsGSTF5 (GenBank Accession No. $\underline{AF309382}$) sequences. The cDNA was composed of a 669-bp open reading frame encoding for 223 amino acids. The deduced peptide of this gene shared on overall identity of 75% with other known phi class GST sequences. On the other hands, the OsGSTF5 sequence showed only 34% identity with the sequence of the OsGSTF3 cloned by our previous study (Cho et al., 2005). This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH-Sepharose affinity column chromatography. The expressed OsGSTF5 formed a homo-dimer composed of 28 kDa subunit and its pI value was approximately 7.8. The expressed OsGSTF5 displayed glutathione conjugation activity toward 1-chloro-2,4-dinitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane and glutathione peroxidase activity toward cumene hydroperoxide. The OsGSTF5 also had high activities towards the herbicides alachlor, atrazine and metolachlor. The OsGSTF5 was highly sensitive to inhibition by S-hexylGSH, benastatin A and hematin. We propose from these results that the expressed OsGSTF5 is a phi class GST and appears to play a role in the conjugation of herbicide and GPOX activity.

• Journal of Periodontal and Implant Science
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• v.38 no.sup2
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• pp.317-324
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• 2008

Purpose: Aggregatibacter actinomycetemcomitans was associated with localized aggressive periodontitis, endocarditis, meningitis, and osteomyelitis. The cytolethal distending toxin (CDT) of A. actinomycetemcomitans was considered as a key factor of these diseases is composed of five open reading frames (ORFs). Among of them, An enzymatic subunit of the CDT, CdtB has been known to be internalized into the host cell in order to induce its genotoxic effect. However, CdtB can not be localized in host cytoplasm without the help of a heterodimeric complex consisting of CdtA and CdtC. So, some studies suggested that CdtC functions as a ligand to interact with GM3 ganglioside of host cell surface. The precise role of the CdtC protein in the mechanism of action of the holotoxin is unknown at the present time. The aim of this study was to generate recombinant CdtC proteins expression from A. actinomycetemcomitans, through gene cloning and protein used to investigate the function of Cdt C protein in the bacterial pathogenesis. Materials and Methods: The genomic DNA of A. actinomycetemcomitans Y4 (ATCC29522) was isolated using the genomic DNA extraction kit and used as template to yield cdtC genes by PCR. The amplifed cdtC genes were cloned into T-vector and cloned cdt C gene was then subcloned to pET28a expression vector. The pET28a-cdtC plasmid expressed in BL21 (DE3) Escherichia coli system. Diverse conditons were tested to opitimize the expression and purification of functional CdtC protein in E. coli. Results: In this study we reconstructed CdtC subunit of A. actinomycetemcomitans Y4 and comfirmed the recombinant CdtC expression by SDS-PAGE and Western Blotting. The expression level of the recombinant CdtC was about 2% of total bacterial proteins. Conclusion: The lab condition of procedure for the purification of functionally active recombinant CdtC protein is established. The active recombinant CdtC protein will serve to examine the role of CdtC proteins in the host recognition and enzyme activity of CDT and investigate the pathological process of A. actinomycetemcomitans in periodontal disease.

• Journal of Microbiology and Biotechnology
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• v.28 no.1
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• pp.1-11
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• 2018

An antioxidant peptide derived from Pinctada fucata meat using an Alcalase2.4L enzymatic hydrolysis method (named AOP) and identified by LC-TOF-MS has promising clinical potential for generating cosmetic products that protect skin from sunshine. To date, there have been few published studies investigating the structure-activity relationship in these peptides. To prepare antioxidant peptides better and improve their stability, the design and expression of an antioxidant peptide from Pinctada fucata (named DSAOP) was studied. The peptide contains a common precursor of an expression vector containing an ${\alpha}$-helix tandemly linked according to the BamHI restriction sites. The DNA fragments encoding DSAOP were synthesized and subcloned into the expression vector pET-30a (+), and the peptide was expressed mostly as soluble protein in recombinant Escherichia coli. Meanwhile, the DPPH radical scavenging activity, superoxide radical scavenging activity, and hydroxyl radical scavenging activity of DSAOP $IC_{50}$ values were $0.136{\pm}0.006$, $0.625{\pm}0.025$, and $0.306{\pm}0.015mg/ml$, respectively, with 2-fold higher DPPH radical scavenging activity compared with chemosynthesized AOP (p < 0.05), as well as higher superoxide radical scavenging activity compared with natural AOP (p < 0.05). This preparation method was at the international advanced level. Furthermore, pilot-scale production results showed that DSAOP was expressed successfully in fermenter cultures, which indicated that the design strategy and expression methods would be useful for obtaining substantial amounts of stable peptides at low costs. These results showed that DSAOP produced with recombinant Escherichia coli could be useful in cosmetic skin care products, health foods, and pharmaceuticals.

• Journal of Microbiology and Biotechnology
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• v.29 no.5
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• pp.749-757
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• 2019

Nitrilase is a valuable hydrolase that catalyzes nitriles into carboxylic acid and ammonia. Its applications, however, are severely restricted by the harsh conditions of industrial reaction processes. To solve this problem, a nitrilase from Acidovorax facilis 72W was inserted into an Escherichia coli-Bacillus subtilis shuttle vector for spore surface display. Western blot, enzyme activity measurements and flow cytometric analysis results all indicated a successful spore surface display of the CotB-nit fusion protein. In addition, the optimal catalytic pH value and temperature of the displayed nitrilase were determined to be 7.0 and $50^{\circ}C$, respectively. Moreover, results of reusability tests revealed that 64% of the initial activity of the displayed nitrilase was still retained at the $10^{th}$ cycle. Furthermore, hydrolysis efficiency of upscale production of cyanocarboxylic acid was significantly higher in the displayed nitrilase-treated group than in the free group expressed by E. coli (pET-28a-nit). Generally, the display of A. facilis 72W nitrilase on the spore surface of Bacillus subtilis may be a useful method for immobilization of enzyme and consequent biocatalytic stabilization.

• Korean Chemical Engineering Research
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• v.55 no.3
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• pp.369-378
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• 2017

Vascular endotherial growth factor (VEGF) is a potent mitogen that stimulates vascular permeability and angiogenesis and has a potential in therapeutic applications. An industrial production method that provides high yield as well as purity is needed. Researches for various factors of mild solubilization with combination of ubiquitin fusion protein to increase solubility were carried out as well as by changing pH and denaturant concentration. Usage of pET28-a bacteral expression vector in BL21 (DE3) host cell was capable of producing approximately 14 g/L VEGF fusion protein in 20L fermentor. A purification process consisting of four chromatography steps including refolding and digestion with UBP1 resulted in mild solublization under the conditions of 2M urea and pH 10.0 due to ubiquitin fusion tag protein that increases in solubility of target protein VEGF. High yield of refolding and dimerization could be obtained between two step Ni-affinity chromatography. Multimeric and misfolded proteins and endotoxin were removed by DEAE anion exchange chromatography. Final monomers were removed from dimers by gel filtration chromatography. Characterization analysis of purified dimeric VEGF was performed using SDS-PAGE and RP-HPLC with a purity of 97%.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.4
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• pp.561-566
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• 2014

The complete coding sequence of Wild Argali ISG15 cDNA was generated by rapid amplification of cDNA ends. The ISG15 cDNA was 642 bp with an open reading frame of 474 bp, which encoded a 17.47 kDa protein composed of 157 amino acids. Its amino acid sequence shared 97.9%, 80.8%, 91.4%, 94.3%, 78.3% identity with those of ISG15cDNA from Ovis aries (accession no. NM001009735.1), Capra hircus (accession no. HQ329186.1), Bos taurus (accession no. BC102318.1), Bubalus bubalis (accession no. HM543269.1), and Sus scrofa (accession no. EU647216.1), respectively. The entire coding sequence was inserted into the pET-28a vector and expressed in E. coli. The recombinant protein corresponded to the expected molecular mass of 25 kDa as judged by SDS-PAGE, and it was detected in the bacterial inclusion bodies. The expressed protein could be purified by $Ni^{2+}$ chelate affinity chromatography and the results from the lymphocyte proliferation test showed that the product could stimulate lymphocyte proliferation very well (p<0.05), which further confirmed its biological activity.

• Korean Journal of Microbiology
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• v.43 no.1
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• pp.19-22
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• 2007

Acetohydroxyacid synthase (E.C.2.2.1.6., AHAS) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine. The AHAS gene (TIGR access code HI2585) from Heamophilus influenzae was cloned into the bacterial expression vector pET-28a and expressed in the Escherichia coli strain BL21(DE3). The expressed enzyme was purified by $Ni^{2+}-charged$ HiTrap chelating HP column. The purified enzyme appears as a single band on SDS-PAGE with a molecular mass of about 63.9 kDa. The enzyme exhibits absolute dependence on the three cofactors FAD, $MgCl_{2}$ and thiamine diphosphate for activity. Specific activity of purified enzyme has 3.22 unit/mg and optimum activity in the pH 7.5 at $37^{\circ}C$. This enzyme activity has an effect on the buffer. When comparing the enzyme activity against the organic solvent, it followed in type and the difference it is but even from the aqueous solution where the organic solvent is included with the fact that the enzyme activity is maintained.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.534-537
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• 2002

An agarase gene from Bacillus cereus ASK202 was expressed in high levels by E. coli BL21(DE3)/pEBA1 using pET28a(+) vector system with the inducible T7 promoter in the presence of isopropyl- ${\beta}$ -thiogalactopyranoside. The open reading frame encodes 761 amino acid residues with a calculated molecular weight of 83,300 daltons and a potential signal peptide about 36 amino acid residues at the N-terminus. E. coli BL21(DE3)/pEBA1 produce 1280 unit/ ${\ell}$ of agarase. The optimum physical condition for the agarase activity was pH 5.6, and $40^{\circ}C$, respectively. The agarase activity was stable up pH $4.0{\sim}9.0$ and $4{\sim}40^{\circ}C$. The km and maximum rate of metabolism for agar were 0.068mg/$m{\ell}$ and 0.094mg/$m{\ell}{\cdot}min$, respectively.

• Microbiology and Biotechnology Letters
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• v.46 no.1
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• pp.51-58
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• 2018

The expression, purification, and characterization of cold-adapted lipase from the psychrophile, Janthinobacterium sp. were investigated. The gene encoding lipase from Janthinobacterium sp. PAMC 25641 was cloned into a pET28a(+) vector and heterologously expressed in Escherichia coli BL21 (DE3). The amino acid sequence deduced from the nucleotide sequence (930 bp) corresponded to a protein having 309 amino acid residues with a molecular weight of 32.7 kDa and a pI of 5.55. Recombinant E. coli harboring the Janthinobacterium lipase gene were induced by addition of isopropyl-${\beta}$-D-thiogalactopyranoside. $Ni^{2+}$-NTA affinity chromatography was used to purify the lipase, which had a specific activity of 107.9 U/mg protein. The effect of temperature and pH on the activity of lipase was measured using p-nitrophenyl octanoate as a substrate. The stability of the lipase at low temperatures indicated it is a cold-adapted enzyme. The lipase activity was increased by $Na^{2+}$, $Mg^{2+}$, and $Mn^{2+}$, and decreased by $Zn^{2+}$ and $Co^{2+}$. Analysis of the lipase activity using various p-nitrophenyl esters showed a strong preference toward short acyl chains of the esters, indicating the ability of the cold-adapted lipase to hydrolyze short-chain esters.