• Korean Journal of Agricultural Science
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• v.41 no.3
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• pp.245-249
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• 2014

Among dozens of DNA polymerases cloned from thermophilic bacteria, Taq DNA polymerase from Thermus aquaticus has been most frequently used in polymerase chain reaction (PCR) that is being applied to gene cloning, DNA sequencing, gene expression analysis, and detection of infectious and genetic diseases. Since native Taq DNA polymerase is expressed at low level in T. aquaticus, recombinant Escherichia coli system was used to produce Taq DNA polymerase in a large amount. Taq DNA polymerase was expressed as a soluble form under the control of tac promoter in E. coli, and purified by heat treatment and ion exchange chromatographies. The purified Taq DNA polymerase was nearly homogeneous and exhibited a similar DNA amplification activity with a commercial Taq DNA polymerase.

• Journal of Marine Bioscience and Biotechnology
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• v.1 no.2
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• pp.119-125
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• 2006

To understand the comprehensive mechanisms of biological function for insulin-like growth factor-I (IGF-I) in vertebrates, we have investigated the cDNA sequence of this gene in the korean rockfish (Sebastes schlegeli). The mature form of korean rockfish IGF-I was found to be comprised of 67 amino acid residues, showing about a 7 kDa molecular weight. In this study, we used the polymerase chain reaction (PCR) to obtain a korean rockfish IGF-I (KR IGF-I) cDNA fragment, and methods of rapid amplification of cDNA ends (RACE) to obtain a full length of the KR IGF-I sequence. The KR IGF-I encoded for a predicted amino acid sequence showed identities of 93.6 %, 90.7 %, and 85.4 % in comparison with flounder, chinook salmon, and human IGF-I, respectively. To obtain recombinant biologically active polypeptides, korean rockfish B-C-A-D domains were amplified using the PCR, then the isolated cDNA was expressed in the E. coli BL21(DE3). The recombinant KR IGF-I protein biological function was measured by stimulation of [$^3H$] thymidine incorporation, suggesting the cDNA codes for the korean rockfish proIGF-I.

• Molecules and Cells
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• v.28 no.1
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• pp.19-24
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• 2009

The goal of this study was to create a novel baculovirus expression system that does not require recombinant virus purification steps. Transfection of insect cells with transfer vectors containing barnase under control of the Cotesia plutellae bracovirus (CpBV) promoters ORF3004 or ORF3005 reduced cell growth. Co-transfection with bApGOZA DNA yielded no recombinant viruses and nonrecombinant backgrounds. To further investigate the detrimental effects of barnase on insect cells, two recombinant bacmids harboring the barnase gene under control of the CpBV promoters, namely bAcFast-3004ProBarnase and bAcFast-3005ProBarnase, were constructed. While no viral replication was observed when only the recombinant bacmids were transfected, recombinant viruses were generated when the bacmids were co-transfected with the transfer vector, pAcUWPolh, through substitution of the barnase gene with the native polyhedrin gene by homologous recombination. Moreover, no non-recombinant backgrounds were detected from unpurified recombinant stocks using PCR analysis. These results indicate that CpBV promoters can be used to improve baculovirus expression vectors by means of lethal gene expression under the control of these promoters.

• Journal of Microbiology
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• v.35 no.4
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• pp.341-346
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• 1997

In order to express recombinant porcine TGF-${\beta}1$ protein in a baculovirus expression system the entire TGF-${\beta}1$gene containing extra amino acids at the N terminus was cloned into pFBa and pFBb of the Bac-To-$Bac^{TM}$ baculovirus expression system. One of the clones contained 106 extra amino acids and was designated pFBa-106 TGF-${\beta}1$, and the other had 28 extra amino acids and was designated pFBb-28 TGF-${\beta}1$. The orientation of the gene was identified with restriction enzyme mapping and PCR with internal TGF-${\beta}1$ primers. Sf-9 cells were infected at a m.o.i. of 10 by the recombinant viruses generated from the two expected sizes of 55 kD and 46.4kD. these precursor forms of TGF-${\beta}1$ with a polyclonal antibody against human TGF-${\beta}1$. No mature form of TGF-${\beta}1$ protein was detected on SDS gels and an immunoblot indicated that TGF-${\beta}1$ precursor is not properlu processed in insect cells.

• Journal of Applied Biological Chemistry
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• v.44 no.4
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• pp.167-172
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• 2001

A cDNA fragment encoding the chloroplastic fructose-1, 6-bisphosphatase (FBPase) was cloned via PCR from the cDNA library of pea leaves. The cloned cDNA, about 1.05 kbp without signal sequence, was introduced into a pET-28a vector for expression in E. coli strain BL21(DE3)pLysS. The recombinant FBPase was purified through $Ni^+-NTA$ affinity chromatography and characterized. Molecular mass of the monomer was about 42,000. Enzymatic activity of the purified enzyme as the native pea chloroplastic FBPase was the highest at alkaline pH (pH 9.0). The recombinant enzyme was activated by a reducing agent DTT and was insensitive to AMP. The activation energy (Ea) and Arrehenius frequency factor were 42.67 kcal/mol and $2.65{\times}10^{14}/s$, respectively, slightly higher than those of the native enzyme. $K_M$ and $V_{max}$ were $99.98{\mu}M$ and $52.9{\mu}M/min$, respectively, which were comparable with the native enzyme.

• Food Science of Animal Resources
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• v.38 no.4
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• pp.816-822
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• 2018

Bifidobacterium is recognized as one of the most beneficial microorganisms in our gut. Many researches on bifidobacteria have been done to understand their roles in the gut. The objective of the present study was to develop a bioluminescent labelling plasmid vector for bifidobacteria to facilitate their visualization in vitro, in situ, and in vivo. A plasmid replicon (2.0 kb) of plasmid pFI2576 previously identified from B. longum FI10564 was amplified by PCR and cloned into pUC19 plasmid vector (2.68 kb). The cloned replicon was subcloned into pTG262 ($luc^+$) recombinant plasmid vector (7.4 kb) where a luciferase gene ($luc^+$) from pLuc2 (8.5 kb), an Escherichia coli and lactobacilli shuttle vector, was inserted into pTG262 plasmid vector. The final recombinant DNA, pTG262::pFI2576 rep ($luc^+$), was transferred into a B. catenulatum strain. This recombinant strain showed 3,024 relative luminescence units at $OD_{600}$ value of 0.352. Thus, this recombinant plasmid construct can be broadly used for labelling bifidobacteria.

• Journal of Fisheries and Marine Sciences Education
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• v.27 no.5
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• pp.1470-1478
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• 2015

Adjuvant is an immune enhancer commonly used during vaccination to enhance the host immune response. In the present study, we produced the several recombinant protein from immune related gene of olive flounder (Paralichthys olivaceus). Especially, to produce the soluble type of recombinant protein, we constructed the MBP (Maltose binding protein) fusion G-CSF (Granulocyte colony stimulating factor) recombinant protein among the flounder immune related genes. To verify the immune stimulatory effect and safety of this recombinant protein (rPoGCSF), expression changes of several immune genes were tested using quantitative real-time PCR method with gene specific primer from flounder head kidney leukocytes. As a result, we confirmed that the rPoGCSF has an ability of immune stimulatory effect, also it has broad range of pH and temperature.

• Preventive Nutrition and Food Science
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• v.15 no.4
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• pp.360-363
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• 2010

To enhance the expression and secretion of pediocin PA-1 from heterologous bacterial hosts, the promoter and deduced signal sequence (PS) of an $\alpha$-amylase gene from a Bifidobacterium adolescentis strain was fused with pediocin PA-1 structural and immunity genes (AB) and the resulting functions were evaluated in Escherichia coli. Two recombinant PCR products were created-one with just the deduced signal sequence and one with the sequence plus the Ser and Thr sequences that are the next two amino acids of the signal sequence. These two products, the PSAB (---AQA::KYY---) and PSABST (---AQA$\underline{ST}$::KYY---), respectively, were inserted into a TA cloning vector (yT&A) and named pPSAB, which was previously reported, and pPSABST. The two recombinant plasmid DNAs were transferred into E. coli JM109 and the transformants displayed antimicrobial activity, where the activity of E. coli JM109 (pPSAB) was stronger than that of E. coli JM109 (pPSABST), indicating that the ST amino acid residues were not necessary for secretion and might have even decreased the antimicrobial activity of recombinant pediocin PA-1.

• Journal of Microbiology
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• v.37 no.4
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• pp.234-242
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• 1999

To obtain the recombinant circumsporozoite (CS) protein for the diagnosis of patients and seroepidemiology of Plasmodium vivax malaria which have been prevalent in northern part of Kyonggido, the CS protein gene was amplified by the polymerase chain reaction (PCR) from genomic DNA of the Korean vivax malaria patient. The gene consists of 1,123 nucleotides except signal peptide sequences and had an uninterrupted reading frame encoding a protein of 374 amino acids with a central region of 20 tandem repeats of the nonapeptide. The CS protein gene was expressed in Escherichia coli and purified, the molecular weight of recombinant CS protein was about 44 kDa (monomer) under denaturing purification and about 65 kDa (dimer) under native purification by SDS-PAGE. The purified recombinant CS protein which has antigenicity to malaria patients in Western blot analysis and Enzyme-linked immunosorbent assay, reacted only with the serum of P. vivax (PV210) infected malaria patients with no cross reaction to the P. falciparum malaria patient. The recombinant CS protein purified in this study will serve as a useful antigen to support the diagnosis of malaria patients and seroepidemiology.

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.886-887
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• 2005