• Journal of Microbiology and Biotechnology
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• 제27권5호
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• pp.983-989
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• 2017

NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of $NADP^+$ in the affinity chromatography process. In the present study, the rat NPR clone containing a $6{\times}$ Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using $Ni^{2+}$-affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochrome c and nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.

• KSBB Journal
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• 제4권3호
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• pp.266-270
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• 1989

실험실 규모의 배양기에서 곤충세포의 배양을 수행하였다. 회분식 배양에서의 곤충 세포의 성장은 serum의 농도, 다른 영양소, 초기 정종 농도, 기계적인 교반파 같은 변수에 의해 영향을 받는 것으로 나타났다. Lactate와 ammonium은 회분식 배양에서의 말기에 관찰되는 농도에서는 세포의 성장을 저해하는 원인은 아닌것 같았다. 또한. redox potential은 공충세포의 배양 용존산소를 측정할 수 있는 좋은 index 임을 알 수 있었다. 아울러 유전공학적으로 재조합된 baculovirus를 곤충세포에 감염시켜 재조합 단백질의 생산을 시도하였으며 dilution rate 가 $0.006\;hr^{-1}$일때 반응기당 최대 2800 units 의 beta-galactosidase 가 생산되었다.

• Journal of Plant Biotechnology
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• 제33권4호
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• pp.233-236
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• 2006

Human serum albumin (HSA) is the most abundant protein in plasma and is the most often used intravenous protein in many human therapies. However, HSA is currently extracted only from plasma because commercially feasible recombinant expression systems are not available. This study attempted to develop an efficient system for recombinant HSA production by chloroplast transformation of tobacco. A HSA cDNA was isolated from a cDNA library constructed with human liver tissue. Chloroplast transformation vectors were constructed by introducing various regulatory elements to HSA regulatory sequences. Vectors were delivered by particle bombardment into leaf explants and chloroplast-transformed plants were subsequently regenerated into whole plants. Southern blot analysis confirmed that the HSA cDNA was incorporated between rps12 and orf70B of the chloroplast genome as designed. Western blot analysis revealed that hyper-expression and increasing the stability of HSA were achieved by modification of the regulatory sequences using the psbA5'UTRs in combination with elements of the 14 N-terminal amino acids of the GFP and the FLAG tag. However, only plants transformed with the vector containing all of these elements were able to accumulate HSA.

• 대한의생명과학회지
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• 제11권1호
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• pp.57-61
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• 2005

The number of sites at which a protein can be readily cleaved by a proteolytic enzyme is greatly influenced by its three-dimensional structure. For native, properly-folded proteins both the rate of cleavage and number of sites at which cleavage takes place are usually much less than for the denatured protein. In order to compare the tertiary structure of recombinant HepG2 type glucose transporter with that of its native counterpart in the erythrocyte, the pattern of tryptic cleavage of the protein expressed in insect cell membranes was therefore examined. After 30 minutes digestion, a fragment of approximate Mr 19,000-21,000 was generated. In addition to this, there were two less intensely stained fragments of apparent Mr 28,000 and 17,000. The pattern of labelling was similar up to 2 hours of digestion. However, the fragments of Mr 19,000-21,000 and Mr 17,000 were no longer detectable after 4 hours digestion. The observation of a very similar pattern of fragments yielded by tryptic digestion of the HepG2 type transporter expressed in insect cells suggests that the recombinant protein exhibits a tertiary structure similar if not identical to that of its human counterpart. Also, the endogenous sugar transporter(s) present in Sf21 cells did not bind cytochalasin B, the potent transporter inhibitor. Therefore, the baculovirus/Spodoptera frugiperda (Sf) cell expression system could be very useful for production of large amounts of human glucose transporters, heterologously.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.232-234
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• 2002

공업적으로 중요성이 날로 더해가는 phospholipase C (PLC) 를 Bacillus cereus를 이용하여 생산하였다. 또한, plc::gfp fusion protein 을 생산하는 재조합 E. coli 를 제조하고 배양하였으며 특히 형광센서를 이용하여 PLC 의 생산 특성을 모니터링하였다.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2006년도 제47회 학술심포지움 및 추계국제학술대회
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• pp.79.2-79.2
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• 2006

• Journal of Microbiology and Biotechnology
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• 제26권4호
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• pp.710-716
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• 2016

Gamma-aminobutyric acid (GABA) is a non-protein amino acid, which is an important inhibitor of neurotransmission in the human brain. GABA is also used as the precursor of biopolymer Nylon-4 production. In this study, the carbon flux from the tricarboxylic acid cycle was directed to the GABA shunt pathway for the production of GABA from glucose. The GABA shunt enzymes succinate-semialdehyde dehydrogenase (GabD) and GABA aminotransferase (GabT) were co-localized along with the GABA transporter (GadC) by using a synthetic scaffold complex. The co-localized enzyme scaffold complex produced 0.71 g/l of GABA from 10 g/l of glucose. Inactivation of competing metabolic pathways in mutant E. coli strains XBM1 and XBM6 increased GABA production 13% to reach 0.80 g/l GABA by the enzymes co-localized and expressed in the mutant strains. The recombinant E. coli system developed in this study demonstrated the possibility of the pathway of the GABA shunt as a novel GABA production pathway.

• 한국미생물·생명공학회지
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• 제23권4호
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• pp.446-453
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• 1995

$\alpha $-Arabinofuranosidase was produced by E. coli HB101 haboring the recombinant plasmid pKMG11 which contained the arfI gene of Bacillus stearothermophilus. The maximum production of the enzyme was observed when E. coli HB101 cells were grown at 37$\circ$C for 20 hours in the medium containing 0.5% arabinose, 1.0% tryptone, 0.5% yeast extract, and 1% NaCl. The $\ALPHA $-arabinofuranosidase produced was purified to homogeneity using a combination of 20-50% ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion exchange column chromatography and Sepharose 6B-100 gel filtration. The purified enzyme was most active at 55$\circ$C and pH 6.5. The K$_{m}$ and V$_{max}$ values of the enzyme on $\rho $-nitrophenyl-$\alpha $-arabinofuranoside was determined to be 2.99 mM and 0.43 $\mu $mole/min (319.74 $\mu $mole/min/mg), respectively. The pI value was 4.5. The molecular weight of the native protein was estimated to be 289 kDa. The SDS-polyacrylamide gel clectrophoresis analysis suggested that the functional protein was a trimer of the 108 kDa identical subunits. The N-terminal amino acid sequence of the a-arabinofuranosidase was identified as X-Ser-Thr-Ala-Pro-Arg( \ulcorner )-Ala-Thr-Met-Val-Ile-Asp-X-Ala-Phe.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권4호
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• pp.304-308
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• 2005

The response of IPTG induction was investigated through the monitoring of the alkali consumption rate and buffer capacity during the cultivation of recombinant E. coli BL21 (DE3) harboring the plasmid pRSET-LacZ under the control of lac promoter. The rate of alkali consumption increased along with cell growth, but declined suddenly after approximately 0.2 h of IPTG induction. The buffer capacity also declined after 0.9 h of IPTG induction. The profile of buffer capacity seems to correlate with the level of acetate production. The IPTG response was monitored only when introduced into the mid-exponential phase of bacterial cell growth. The minimum concentration of IPTG for induction, which was found out to be 0.1 mM, can also be monitored on-line and in-situ. Therefore, the on-line monitoring of alkali consumption rate and buffer capacity can be an indicator of the metabolic shift initiated by IPTG supplement, as well as for the physiological state of cell growth.

• 한국고분자학회:학술대회논문집
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• 한국고분자학회 2006년도 IUPAC International Symposium on Advanced Polymers for Emerging Technologies
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• pp.256-256
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• 2006

The design and synthesis of novel biomolecular materials, based on mimicking the properties of molecules found in nature, is providing materials with unusual properties. Resilin serves as an energy storage material in insects and facilitates flight, jumping (in fleas, froghoppers etc) and sound production (cicadas, etc). Resilin is initially produced as a soluble protein and in its mature form is crosslinked through formation of dityrosine units into a very large insoluble polymer. In the present study, we have synthesized a recombinant form of resilin that can be photochemically cross-linked into a resilient, rubber-like biomaterial that may be suitable for spinal disc implants. This material is almost perfectly elastic and its fatigue lifetime in insects must be >500 million cycles.