• 한국미생물·생명공학회지
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• 제46권3호
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• pp.244-252
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• 2018

Protein disulfide isomerase A3 (PDIA3) is a major member of the protein disulfide isomerase (PDI) family. PDI proteins commonly reside in the endoplasmic reticulum and mediate important thiol-disulfide interchanges during post-translational protein folding. Unlike other PDI family members, PDIA3 is ubiquitous in various organ systems. However, its physiological activity varies in other tissues. PDIA3 has been associated with cancer, airway inflammation, neurodegenerative diseases, and metabolic diseases. However, the mechanisms of the association of PDIA3 with these pathological conditions remain unclear. Recombinant PDIA3 (rPDIA3) is needed to clarify the interactions between PDIA3 and certain physiological phenomena. In the present study, we aimed to produce highly purified rPDIA3 for use in pathological experiments. We expressed rPDIA3 with a histidine-enriched elongated peptide tag in Escherichia coli and obtained rPDIA3 at 97.8% purity using consecutive His-tag and reverse-phase chromatography. Elongated peptide tags screened from artificially designated library had dual functions for protein expression and simple purification.

• KSBB Journal
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• 제32권1호
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• pp.16-21
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• 2017

The multiple sclerosis caused by multiple inflammatory disease or immune system disorder, is usually treated by interferon ${\beta}$ through adjusting the abnormal immune reactions. For high production of human interferon ${\beta}$ using recombinant E. coli, codon optimized and wild type genes were synthesized. When pET-15b or pET-21a vector was used as an expression vector with each gene, there was no target protein expression. When pQE30 vector was used as an expression vector, human interferon ${\beta}$ was expressed by recombinant E. coli XL1-blue and E. coli JM109. Using the codon optimized gene, the expression of human interferon ${\beta}$ was slightly increased as compared to that from wild type gene. However, most of expressed human interferon ${\beta}$ was insoluble form.

• Journal of Ginseng Research
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• 제36권4호
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• pp.418-424
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• 2012

This study focused on the enzymatic biotransformation of the major ginsenoside Rb1 into Rd for the mass production of minor ginsenosides using a novel recombinant ${\beta}$-glucosidase from Flavobacterium johnsoniae. The gene (bglF3) consisting of 2,235 bp (744 amino acid residues) was cloned and the recombinant enzyme overexpressed in Escherichia coli BL21(DE3) was characterized. This enzyme could transform ginsenoside Rb1 and gypenoside XVII to the ginsenosides Rd and F2, respectively. The glutathione S-transferase (GST) fused BglF3 was purified with GST-bind agarose resin and characterized. The kinetic parameters for ${\beta}$-glucosidase had apparent $K_m$ values of $0.91{\pm}0.02$ and $2.84{\pm}0.05$ mM and $V_{max}$ values of $5.75{\pm}0.12$ and $0.71{\pm}0.01{\mu}mol{\cdot}min^{-1}{\cdot}mg$ of $protein^{-1}$ against p-nitrophenyl-${\beta}$-D-glucopyranoside and Rb1, respectively. At optimal conditions of pH 6.0 and $37^{\circ}C$, BglF3 could only hydrolyze the outer glucose moiety of ginsenoside Rb1 and gypenoside XVII at the C-20 position of aglycon into ginsenosides Rd and F2, respectively. These results indicate that the recombinant BglF3 could be useful for the mass production of ginsenosides Rd and F2 in the pharmaceutical or cosmetic industry.

• Biomolecules & Therapeutics
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• 제12권3호
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• pp.179-184
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• 2004

The FimH subunit of type 1-fimbriated Escherichia coli has been determined as a major cause for urinary tract infections. In our previous study, the Adhesin/CTXA2B was expressed as soluble recombinant chimaeric protein derived from the uropathogenic Escherichia coli adhesin genetically coupled to cholera toxin A2B (CTXA2B) subunit in Escherichia coli. Since it is very important to optimize IPTG concentration and culture temperature to maximize cell growth and productivity, These optimal culture factors were determined to increase the productivity of the expressed Adhesin/CTXA2B chimaeric protein in Escherichia coli TB1 carrying pMALfimH/ctxa2b. Our data demonstrate that optimal concentration of IPTG for increased production of chimaeric protein was 0.5 mM. Additionally, culture time was 10 hours and temperature, 37${\circ}C$.

• 생명과학회지
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• 제19권5호
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• pp.561-565
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• 2009

인간조직인자는 혈액응고인자 factor VII 과 복합체를 형성하며 연속적인 혈액응고 연쇄반응을 촉매하는 효소 활성체이다. 복합체 형성에 필수적인 이 조직인자의 세포 외 부분이, 기존의 융합 단백질 및 히스티딘 말단이 없는 새로운 발현 벡터에 의해 대장균 내에서 과량 발현 되었다. 봉입체 형태로 발현된 재조합 인간조직인자는 DEAE-Sephacel 크로마토그라피 기술을 적용하여 분리, 정제 및 구조적 복원이 동시에 시도 되었다. 정제된 재조합 단백질은 SDS-PAGE 분석에서 순수한 형태로 나타났으며, 생물학적 활성도 또한 기존의 조직인자와 거의 동등함을 보였다. 본 연구의 발현 및 정제 시스템은 이전의 보고에서 보여진 방법들에 비해 단백질 분해효소를 사용하지 않아 추가적인 크로마토그라피 과정이 필요 없어 좀 더 효율적이기 때문에 기존의 발현 시스템에 대해 대체할 수 있는 매우 유용한 방법으로 제공된다.

• Asian-Australasian Journal of Animal Sciences
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• 제23권3호
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• pp.401-409
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• 2010

FLICE inhibitory protein (FLIP) is one of the important anti-apoptotic proteins in the Fas/FasL apoptotic path which has death effect domains, mimicking the pro-domain of procaspase-8. To reveal the intracellular signal transduction molecules involved in the process of follicular development in the bovine ovary, we cloned the c-FLIP(L) gene in bovine ovary tissue with the reverse transcription polymerase chain reaction (RT-PCR), deleted the termination codon in its cDNA, and directionally cloned the amplified c-FLIP(L) gene into eukaryotic expression vector pAcGFP-Nl, including AcGFP, and successfully constructed the fusion protein recombinant plasmid. After identifying by restrictive enzyme BglII/EcoRI and sequencing, pAcGFP-bFLIP(L) was then transfected into follicular granulosa cells, mediated by Lipofectamine 2000, the expression of AcGFP observed and the transcription and expression of c-FLIP(L) detected by RT-PCR and Western blot. The results showed that the cattle c-FLIP(L) was successfully cloned; the pAcGFPbFLIP(L) fusion protein recombinant plasmid was successfuly constructed by introducing a BglII/EcoRI cloning site at the two ends of the c-FLIP(L) open reading frame and inserting a Kozak sequence before the start codon. AcGFP expression was detected as early as 24 h after transfection. The percentage of AcGFP positive cells reached about 65% after 24 h. A 1,483 bp transcription was amplified by RT-PCR, and a 83 kD target protein was detected by Western blot. Construction of the pAcGFP-bFLIP(L) recombinant plasmid should be helpful for further understanding the mechanism of regulation of c-FLIP(L) on bovine oocyte formation and development.

• 생명과학회지
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• 제20권11호
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• pp.1577-1581
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• 2010

조혈촉진 cytokine인 Granulocyte colony stimulating factor (G-CSF)는 골수세포를 자극하여 granulocyte로 증식, 분화시키는 기능을 가지며, 현재 아주 고가의 치료제로 사용되고 있다. 인간 G-CSF (hG-CSF)를 아직 시도되지 않은 누에 유래 세포주인 BM5 세포에서 발현시키고 생산 효율을 높이기 위해 hG-CSF cDNA를 변형하였다. hG-CSF의 cDNA의 endoplasmic reticulum (ER) signal sequence 부분을 누에의 소포체에서 분비되는 단백질인 prophenoloxidase (PPAE), protein disulfide isomerase (PDI)와 bombyxin (BX)에서 유래한 누에특이 ER signal sequence로 대체한 hG-CSF의 cDNA 함유 벡터를 구축하였다. 이들 벡터를 사용하여 형질전환한 BM5 세포의 배양액에 분비된 G-CSF 단백질을 western blot으로 분석하여 발현을 확인하였다. 누에특이 ER signal sequence들로 대체된 hG-CSF cDNA를 포함하는 벡터에 의한 hG-CSF 단백질 생산이 인간 G-CSF cDNA가 든 벡터에 의한 hG-CSF의 생산보다 월등히 효율적이었다. 또한, PPAE-signal sequence를 포함하는 hG-CSF 단백질은 배양배지에서 형질전환 4일 후에 최고에 달하였고, 7 일째까지 비슷한 양이 배지 내에서 검출되었다. 이상의 결과는 인간유래 유전자가 곤충세포 내에서 발현 될 때 인간유래 유전자 보다는 곤충 유전자발현 시스템에 맞게 변형했을 경우 더 효율적인 단백질 발현을 얻을 수 있음을 보여 준다.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2002년도 추계학술대회
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• pp.121-124
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• 2002

This work was initiated to develope a recombinant oral poliovaccine (OPV), which is highly advanced in safety (minimizing VAPP) by introducing Type 2,3 poliovirus epitopes into our RPS-Vax system. We have introduced several potential vaccine epitopes of poliovirus Type 2, and 3 into RPS-Vax system, resulting in production of recombinant polioviruses. Any of these chimeric viruses, however, were not detected for their foreign gene expression by serotype-specific mouse antiserum. We have designed several folding units to stabilize the introduced vaccine protein and attached short epitope-concatamer or epitope-multimer to them, followed by production of chimeric viruses. Only those who have an HIV-1 Tat-mediated folding unit were nicely detected for the introduced foreign proteins by anti-Tat antiserum and type-specific peptide-induced antisera. Nevertheless, introduced epitopes were not detected in Western blot experiment with each serotype-specific antiserum. None of the mice inoculated with these chimeric viruses showed preventative immunity when challenged with Lansing and Leon wildtype 2 and 3 poliovirus, and the antiserum did not show neutralizing capacity in vitro. Conformational epitope covering B/C loop region of type 2 and 3 were newly designed by computer modeling, and introduced into the RPS-Vax vector system, followed by production of chimeric viruses. Introduced epitope regions were nicely detected by anti-Tag23 mAb or peptide antibody, but still not detected by poliovirus antiserum. Nevertheless, neutralizing antibody was detected in the Tg-PVR mice even when inoculated once with these chimeric viruses. Also, the immunized mice showed perfect preventative immunity against the wild Type poliovirus Lancing or Leon. When boosted appropriately, those chimeric virus-inoculated Tg-PVR mice produced equivalent amounts of neutralizing antibody to those in Sabin 2/3-immunized mice. These data strongly suggest that our recombinant poliovirus (RPS-PV2 and RPS-PV3) can be used as a safe and effective rec-OPV instead of any preexisting poliovaccine.

• Molecules and Cells
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• 제38권4호
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• pp.318-326
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• 2015

We previously reported that the SbROMT3syn recombinant protein catalyzes the production of the methylated resveratrol derivatives pinostilbene and pterostilbene by methylating substrate resveratrol in recombinant E. coli. To further study the production of stilbene compounds in E. coli by the expression of enzymes involved in stilbene biosynthesis, we isolated three stilbene synthase (STS) genes from rhubarb, peanut, and grape as well as two resveratrol O-methyltransferase (ROMT) genes from grape and sorghum. The ability of RpSTS to produce resveratrol in recombinant E. coli was compared with other AhSTS and VrSTS genes. Out of three STS, only AhSTS was able to produce resveratrol from p-coumaric acid. Thus, to improve the solubility of RpSTS, VrROMT, and SbROMT3 in E. coli, we synthesized the RpSTS, VrROMT and SbROMT3 genes following codon-optimization and expressed one or both genes together with the cinnamate/4-coumarate:coenzyme A ligase (CCL) gene from Streptomyces coelicolor. Our HPLC and LC-MS analyses showed that recombinant E. coli expressing both ScCCL and RpSTSsyn led to the production of resveratrol when p-coumaric acid was used as the precursor. In addition, incorporation of SbROMT3syn in recombinant E. coli cells produced resveratrol and its mono-methylated derivative, pinostilbene, as the major products from p-coumaric acid. However, very small amounts of pterostilbene were only detectable in the recombinant E. coli cells expressing the ScCCL, RpSTSsyn and SbROMT3syn genes. These results suggest that RpSTSsyn exhibits an enhanced enzyme activity to produce resveratrol and SbROMT3syn catalyzes the methylation of resveratrol to produce pinostilbene in E. coli cells.

• Asian-Australasian Journal of Animal Sciences
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• 제14권1호
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• pp.17-20
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• 2001

The effect of chicken IGF-I on protein synthesis of chicken embryo myoblasts cultured in serum-free medium was examined. When myoblasts were expanded to approximate 20-30% of well, the medium was changed to the serum-free medium including 0, 2, 20, 200 or 2000 ng/ml of recombinant chicken IGF-I. The culture medium including 10% fetal calf serum (FCS) was used as positive control. After 1 day of incubation, protein synthesis was measured by the incorporation of [$^3H$]-L-leucine. Thereafter cells were continued to incubate for further 18 hours, and the radioactivity in the protein was measured as an index of protein synthesis. The values for protein synthesis cultured in the serum-free medium without chicken IGF-I or with 2000 ng/ml of chicken IGF-I were the lowest. Protein synthesis was elevated with increasing chicken IGF-I concentration from 0 to 20 ng/ml. The values for protein synthesis in the 20 ng/ml and 200 ng/ml IGF-I groups were about half of that of the FCS group. The present study revealed that the potency of chicken IGF-I at the levels of 20 to 200 ng/ml to stimulate myoblast protein synthesis was about half of that of 10% FCS.