• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.467-473
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• 2010

To improve the expression efficiency of recombinant endo-$\beta$-1,4-glucanase in P. pastoris, the endo-$\beta$-1,4-glucanase (egI) gene from Aspergillus niger was synthesized using optimized codons. Fourteen pairs of oligonucleotides with 15 bp overlap were designed and the full-length syn-egI gene was generated by two-step PCR-based DNA synthesis. In the synthesized endo-$\beta$-1,4-glucanase gene syn-egI, 193 nucleotides were changed, and the G+C content was decreased from 54% to 44.2%. The syn-egI gene was inserted into pPIC9K and transformed into P. pastoris GS115 by electroporation. The enzyme activity of recombinant P. pastoris stain 2-7# reached 20.3 U/ml with 1% barley $\beta$-glucan and 3.3 U/ml with 1% carboxymethylcellulose (CMC) as substrates in shake flasks versus 1,270.3 U/ml and 220.7 U/ml for the same substrates in 50-1 fermentors. The molecular mass of the recombinant protein was approximately 40 kDa as determined by SDS-PAGE analysis, the optimal temperature for recombinant enzyme activity was $70^{\circ}C$, and the optimal pH was 5.0 when CMC was used as the substrate.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권6호
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• pp.516-521
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• 2006

The use of gltA gene, as a new biomarker for environmental stress biomonitoring, was investigated because of its key position as the first enzyme of the tricarboxylic acid (TCA) cycle. A recombinant bioluminescent Escherichia coli strain, EBJM2, was constructed using a plasmid carrying the citrate synthase (gltA) promoter transcribing the Photorhabdus luminescens IuxCDABE genes (gltA::luxCDABE). The responses from this strain were studied with five different classes of toxicants: DNA damage chemicals, phenolics, oxidative-stress chemicals, PAHs, and organic solvents. EBJM2 responded strongly to DNA damage chemicals, such as mitomycin C (MMC) and methyl-nitro-nitrosoguanidine (MNNG) and nalidixic acid with the strongest responses. In contrast, tests with several compounds from the other four classes of toxicants gave no significant response. Therefore, EBJM2 was found to be sensitive to DNA damage chemicals.

• Journal of Microbiology and Biotechnology
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• 제2권1호
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• pp.50-55
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• 1992

A genomic library of a mesophilic cellulolytic anaerobe, Clostridium sp. KCTC 8440 DNA was constructed in Escherichia coli using plasmid pUC9. Clones of E. coli exhibiting carboxymethyl cellulose-hydrolyzing activity (CMCase) were isolated and divided into seven types based on the restriction enzyme patterns of recombinant plasmids. E. coli strains carrying type A genes showed activity on carboxymethyl cellulose about 7-8 times greater than clones carrying genes of other types. Restriction maps of the cloned DNA fragments were determined, and homologies between them were investigated. The results suggest that Clostridium sp. KCTC 8440 has seven distinct CMCase genes.

• Journal of Animal Science and Technology
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• 제50권2호
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• pp.177-184
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• 2008

Acid-labile subunit(ALS)는 85-kDa 크기의 당단백질로서 7.5-kDa의 insulin-like growth factor(IGF) 및 40~45-kDa IGF-binding protein-3와 결합하여 150-kDa ternary complex를 형성하는 혈장단백질이다. 선행연구에서 본 연구진은 reverse transcription-polymerase chain reaction(RT-PCR) 방법으로 돼지(porcine) ALS(pALS)의 coding sequence를 합성하여 plasmid vector에 삽입시켜 ‘expression construct’를 제작한 바 있다. 그러나 본 expression construct의 pALS coding sequence에는 PCR error로 추정되는 원인으로 말미암아 2개의 bases에서 mis-sense mutation이 일어난 것이 발견되었다. 본 연구에서는 ‘site-directed mutagenesis’ 방법으로 pALS의 올바른 coding sequence를 합성하여 ‘insert DNA’의 마지막 codon 다음에 ‘His-tag’ sequence가 위치한 pET- 28a(+) plasmid expression vector에 삽입하였다. 본 expression construct는 E. coli BL21(DE3) 세포에서 ‘induction’ 시켰고, 발현된 유전자재조합(recombinant) peptide는 Ni-affinity chromato- graphy로 정제하였다. 이렇게 affinity chro- matography로 정제된 peptide는 SDS-PAGE에서 66kDa 위치에 single band를 나타냄으로써 recombinant pALS의 예상된 질량과 일치하였다. 이상의 결과는 본 연구에서 recombinant pALS peptide가 성공적으로 발현정제되었음을 시사한다.

• Food Science and Biotechnology
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• 제17권2호
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• pp.438-441
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• 2008

A novel lactobacilli replicon from plasmids of Lactobacillus reuteri KCTC 3678 was isolated. Eight L. reuteri strains from Korean Collection for Type Cultures (KCTC) and Korea Food Research Institute (KFRI) were screened for cryptic plasmids and most strains harbored 1 or 2 plasm ids. Particularly, L. reuteri KCTC 3678 contained 6 plasm ids which all were used for screening of lactobacilli replicon. EcoRI digests of the plasmid DNA prep from L. reuteri KCTC 3678 were ligated with pUC19 and the recombinant DNAs were serially named from pLR1 to pLR7. A cat (chloramphenicol acetyltransferase; $Cm^r$) gene originated from pC194 was introduced into pLR1-7, resulting in pLR1cat-pLR7cat, respectively. The recombinant plasmids were introduced into L. reuteri KCTC 3679, and only transformants harboring pLR5cat were obtained, indicating that the insert in pLR5 functioned as a lactobacilli replicon.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.230.3-231
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• 2003

The therapeutic use of protein pharmaceuticals produced by recombinant DNA technology is increasing in recent decades. In order to investigate the quality of recombinant proteins, it is important to identify and assign the impurities produced in the process of recombination or in storage conditions. Capillary Electrophoresis is emerging technology exhibiting high sensitivity, selectivity and speed and may be most powerful tools for this application. In this study, human growth hormone (hGH) has been analyzed by various mode of capillary electrophoresis such as capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), and capillary isoelectric focusing (cIEF) to indicate the chemically or biologically oriented variants and the degraded fragments. (omitted)

• 한국잠사학회:학술대회논문집
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• 한국잠사학회 2003년도 International Symposium of Silkworm/Insect Biotechnology and Annual Meeting of Korea Society of Sericultural Science
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• pp.157-159
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• 2003

We have cloned and sequenced the cDNA coding fur a cellulase from the mulberry longicorn beetle, Apriona germari, with the polymerase chain reaction. And then we have constructed the recombinant plasmid vector for Bombyx mori transfomation experiment. (omitted)

• Journal of Microbiology and Biotechnology
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• 제20권2호
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• pp.265-270
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• 2010

Two major endoglucanase genes (cel7B and cel5A) were cloned from Penicillium decumbens 114-2 using the method of modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The result of Southern blotting suggested that P. decumbens has a single copy of the cel5A gene and a single copy of the cel7B gene in its chromosomal DNA. The expression levels of cel5A and cel7B were determined by means of real-time quantitative PCR, suggesting that the two genes were coordinately expressed, and repressed by glucose and induced by cellulose. Both endoglucanase genes were expressed in Saccharomyces cerevisiae and the recombinant proteins were purified. The recombinant Cel7B and Cel5A were both optimally active at $60^{\circ}C$ and pH 4.0. The recombinant Cel7B showed more than 8-fold, 30-fold, and 5-fold higher enzyme activities toward carboxymethyl cellulose, barley $\beta$-glucan, and PASC, respectively, in comparison with that of Cel5A. However, their activities toward pNPC and Avicel showed minor differences. The results suggested that Cel7B is a strict endoglucanase, whereas Cel5A showed processivity because of its relative higher ability to hydrolyze the crystal cellulose.

• Archives of Pharmacal Research
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• 제19권6호
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• pp.469-474
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• 1996

A rapid and sensitive method has been developed to detect hepatitis B virus DNA (HBV) by nested polymerase chain reaction (PCR) technique with primers specific for the surface and core regions in capillary thermal cycler within 80 min. The lower limit for detection by present PCR method is $10^{-5}$ pg of recombinant HBV DNA which is equivalent to that determined by one round of PCR amplification and Southern blot hybridization analysis. When boiled HBV positive serum was serially diluted 10-fold, HBV DNA was successfully determined in $1{\mu}l-10^{-3}$ of serum. HBV DNA was detected by present method in 69 clinical samples including HBsAg positives and negatives by enzyme-linked immunosorbent assay (ELISA). When serum samples were amplified by nested PCR using surface and core region primers, HBV DNAs were detected in 37 of 69 samples (53.6%) and 18 of 69 samples (26.1%), respectively. These results can inform the infectious state of HBsAg positive pateints. A simple and rapid nested PCR protocol by using boiled serum as DNA template has been described for the clinical utility to determine HBV DNA in human serum.

• 분석과학
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• 제12권6호
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• pp.565-570
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• 1999

Bacillus stearothermopilus의 염색체 DNA로부터 polymerase chain reaction 법을 이용하여 ADH의 구조유전자를 증폭시킨 후, 발현 벡터 pGEX-KC에 삽입시켜 glutathion S-tansferase와 융합 단백질로 대장균에서 대랑 발현시켰다. 재조합 ADH는 $37^{\circ}C$에서 1 mM의 isopropyl-${\beta}$-D-thiogalactopyranoside로 단백질의 발현을 유도시켰으며, 발현된 단백질은 glutathione affinity 컬럼을 이용하여 정제하였다. 재조합 ADH는 에탄올에 높은 기질특이성을 나타내었으며 최적 pH와 온도는 각각 pH 9.0과 $70^{\circ}C$이었다. 또한 이 재조합 ADH는 본래의 효소보다 열에 안정하였다. 이 효소는 알코을 측정을 위한 효소학적 방법과 알코올의 공업적 생산에 이용될 수 있다.