• Journal of Microbiology
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• 제37권2호
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• pp.90-96
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• 1999

The core protein of the hepatitis C virus (HCV) is a multifunctional protein. The HCV core protein was reported to regulate cellular gene expression and transform primary rat embryo fibroblast cells. However, the role of the core protein in the pathogenesis of HCV-associated liver diseases is not well understood. To investigate the functional role of the core protein in cytophathogenicity, we have constructed stable expression systems of full length or truncated HCV core protein lacking the C-terminal hyderophobic domains and established HepG2 cell clones constitutively expressing the core protein. The full length core protein was localized in the cytoplasm and the C-terminal truncated core protein was localized in the nucleus. HepG2 cells expressing nuclear, truncated core protein showed elevated cell death during cultivation compared to untransfected cells and full length core-expressing cells. In the treatment with bleomycin, both cell clones expressing full length or truncated core protein appeared to be more sensitive to blemoycin than the parental HepG2 cells. These results suggest that the core protein may play a role in HCV pathogenesis promoting apoptotic cell death of infected cells.

• KSBB Journal
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• 제21권4호
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• pp.302-305
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• 2006

PCR증폭기술 및 무세포 단백질 발현 기술의 융합을 통하여, 여러 형태로 서열의 일부가 결손된 단백질들을 고속으로 발현할 수 있는 시스템을 구축하였다. Exonuclease 및 endonuclease에 대한 mRNA의 안정성 향상을 통하여, PCR 증폭을 통해 획득한 선형 DNA로부터의 안정적인 단백질 발현이 가능하였다. 동일한 플라스미드로부터 출발하여 수 시간 이내에 C-말단의 아미노산서열이 순차적으로 제거된 다양한 형태의 트랜스아미나제 Vf의 활성변화를 확인할 수 있었으며 이같은 기술은 각종 효소 단백질의 서열-활성 상호관계의 연구를 위한 유용한 기반을 제공할 것으로 기대된다.

• Journal of Microbiology and Biotechnology
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• 제30권12호
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• pp.1810-1818
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• 2020

Inhibitor K562 (IK) protein was first isolated from the culture medium of K562 cells, a leukemia cell line, and is an inhibitory regulator of interferon-γ-induced major histocompatibility complex class II expression. Recently, exogenous truncated IK (tIK) protein showed potential as a therapeutic agent for inflammation-related diseases. In this study, we designed a novel putative anti-inflammatory peptide derived from tIK protein based on homology modeling of the human interleukin-10 (hIL-10) structure, and investigated whether the peptide exerted inhibitory effects against pro-inflammatory cytokines such as IL-17 and tumor necrosis factor-α (TNF-α). The peptide contains key residues involved in binding hIL-10 to the IL-10 receptor, and exerted strong inhibitory effects on IL-17 (43.8%) and TNF-α (50.7%). In addition, we used circular dichroism spectroscopy to confirm that the peptide is usually present in a random coil configuration in aqueous solution. In terms of toxicity, the peptide was found to be biologically safe. The mechanisms by which the short peptide derived from human tIK protein exerts inhibitory effects against IL-17 and TNF-α should be explored further. We also evaluated the feasibility of using this novel peptide in skincare products.

• 생명과학회지
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• 제28권4호
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• pp.478-482
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• 2018

인간 rotavirus는 영아에게 급성 설사를 일으키는 병원체의 하나이다. 본 연구에서는 Escherichia coli의 코돈 선호도를 따라서 인간 rotavirus A (serotype 1 strain WA)의 $VP8^*$ 단백질을 일부분 암호화하도록 인공적인 유전자를 합성하였다. 합성된 $VP8^*$ 유전자는 코돈을 번역틀에 일치시키고 클로닝이 용이하도록 하기 위한 NdeI 및 HindIII 제한효소 절단 부위와 친화적 정제를 위한 6-히스티딘 암호화 서열을 C-말단에 보유하고 있다. 합성된 $VP8^*$ DNA 절편을 pT7-7 발현 벡터에 삽입하여 E. coli BL21 (DE3)로 형질전환한 후에 최종 농도 0.05 mM IPTG로 생산을 유도한 결과 예상했던 대로 19.7-kDa 크기의 $VP8^*$ 단백질이 고농도로 발현되었다. SDS-PAGE에 전개된 단백질들을 대상으로 mouse anti-rotavirus capsid antibody를 사용한 Western blotting의 결과 ~20-kDa $VP8^*$ 단백질 밴드가 관찰되었다. 인공 $Vp8^*$ 단백질이 피하 주사된 토끼의 polyclonal antibody 혈장을 이용한 조사에서도 동일한 크기의 단백질 밴드를 확인할 수 있었다. 이는 합성된 유전자가 바이러스성 질환을 통제할 항원성 백신 후보의 생산 혹은 진단용 항체를 개발하기 위한 쉽고 빠른 방법을 제공할 수 있다는 의미이다.

• Journal of Microbiology and Biotechnology
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• 제12권5호
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• pp.807-812
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• 2002

In order to analyze the cellular proteins which interact with core protein of hepatitis C virus (HCV), a yeast two-hybrid screening technique was employed. A carboxyl terminus truncated core protein, which contained amino acid residues from the 1st to 120th, was used as a bait to screen cellular proteins. The expression library prepared from HeLa cell was screened and 400 positive clones were selected. The 75 clones from the positive clones were sequenced and analyzed by undergoing the Blast search. Interestingly, 7 out of the 75 clones encoded E7 antigen of human papilloma virus (HPV). We studied in detail the Interaction between the truncated version of HCV core and E7 antigen in vitro. The core$_{120}$ protein expressed in chimeric form with G57 was able to bring down the E7 protein of HPV type 18 expressed in bacteria. It is therefore suggested that the core of HCV might affect the interaction between E7 and a normal cellular tumor suppressor, known as Rb protein.

• BMB Reports
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• 제52권5호
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• pp.330-335
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• 2019

Hepatitis B virus (HBV) encoding the HBV x protein (HBx) is a known causative agent of hepatocellular carcinoma (HCC). Its pathogenic activities in HCC include interference with several signaling pathways associated with cell proliferation and apoptosis. Mutant C-terminal-truncated HBx isoforms are frequently found in human HCC and have been shown to enhance proliferation and invasiveness leading to HCC malignancy. We investigated the molecular mechanism of the reduced doxorubicin cytotoxicity by C-terminal truncated HBx. Cells transfected with C-terminal truncated HBx exhibited reduced cytotoxicity to doxorubicin compared to those transfected with full-length HBx. The doxorubicin resistance of cells expressing C-terminal truncated HBx correlated with upregulation of the ATP binding cassette subfamily B member 1(ABCB1) transporter, resulting in the enhanced efflux of doxorubicin. Inhibiting the activity of ABCB1 and silencing ABCB1 expression by small interfering ribonucleic acid (siRNA) increased the cytotoxicity of doxorubicin. These results indicate that elevated ABCB1 expression induced by C-terminal truncation of HBx was responsible for doxorubicin resistance in HCC. Hence, co-treatment with an ABCB1 inhibitor and an anticancer agent may be effective for the treatment of patients with liver cancer containing the C-terminal truncated HBx.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권6호
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• pp.599-602
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• 2005

Truncated form of UBP1, an ubiquitin-specific protease of Saccharomyces cerevisiae, was overexpressed in Escherichia coli. The hexahistidine residue $(His_6)$ was fused to the N-terminus of truncated UBP1 and the corresponding recombinant protein was purified with high yield by immobilized metal affinity chromatography. The truncated form of UBP1 protein was functional to cleave ubiquitinated human growth hormone as substrate. Effects of pH and temperature were investigated in order to optimize deubiquitinating reactions for the truncated UBP1. Optimum temperature and pH for the cleavage reaction were $40^{\circ}C$ and pH 8.0, respectively.

• 대한바이러스학회지
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• 제27권2호
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• pp.105-113
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• 1997

C 형 간염 바이러스 (Hepatitis C Virus, HCV)는 두종류의 외피당단백질 $E1_{192-383}$ 및 $E2_{384-746}$를 갖고 있다. $E1_{192-383}$ 및 $E2_{384-740}$ 단백질은 glutathione S-transferae (GST) 융합단백질의 형태로 대장균에서 발현되지 않았으나, 이 단백질들의 C말단에 존재하는 소수성영역을 제거하였을 때 $GST-E1_{192-283}$ 융합단백질은 과량으로 가용성 형태로 발현되었고, $GST-E2_{384-649}$ 융합단백질은 비 가용성 형태로 발현되었다. 이 융합단백질들 각각은 HCV 양성환자의 혈청과 특이적으로 반응하였다. Thrombin으로 처리하여 얻은 정제된 $E1_{192-283}$ 단백질 및 융합형태의 $GST-E2_{384-649}$ 단백질 각각을 생쥐에 접종하였을 때 E1 및 E2 특이적인 항체가 생성되었다. 이 결과들은 $E1_{192-383}$ 및 $E2_{384-649}$ 융합단백질 C 말단에 존재하는 소수성영역이 이 단백질들의 발현량 및 가용성에 영향을 주며 $E1_{192-283}$ 단백질 영역내에 HCV 양성환자의 혈청과 특이적으로 반응하는 epitope (s)이 존재한다는 것을 제시해 주고 있다.

• 대한미생물학회지
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• 제35권4호
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• pp.273-282
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• 2000

dTDP-rhamnose provides L-rhamnose to the bridge-like structure between mycolyl arabinogalactan and peptidoglycan of the mycobacterial cell wall. dTDP-rhamnose is composed of glucose-l-phosphate and dTTP by four enzymes encoded by rmlA-D. To determine the region(s) of RmlC protein essential for its dTDP-4-keto-6-deoxyglucose epimerase activity, we overexpressed both whole (202 amino acids) and three different truncated (N-terminal 106 or 150 or C-terminal 97 amino acids) RmlC proteins of Mycobacterium tuberculosis. The RmlC enzyme activity in the soluble lysates of ${\Delta}rmlC$ E. coli strain $S{\Phi}874$ (DE3 PlysS) expressing the wild type or truncated rmlC genes was initially analyzed by three sequential reactions from dTDP-glucose to dTDP-rhamnose in the presence of purified RmlB and RmlD. All three soluble lysates containing the truncated RmlC proteins showed no enzyme activity, while that containing the wild type RmlC was active. This wild type RmlC was then overexpressed and purified. The incubation of the purified RmlC enzyme so obtained with dTDP-4-keto-6-deoxyglucose resulted in the conversion of dTDP-4-keto-rhamnose. The results show that the truncated regions of the RmlC protein are important for the RmlC enzyme activity in M. tuberculosis.

• Journal of Microbiology
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• 제33권2호
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• pp.165-170
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• 1995

The 356 amino acid long lambda integrase protein of bacteriophage .lambda. constains two autonomous DNA binding domains with distinct sequence specificities. The amino terminal domain of integrase is implicated to bind to the arm-type sequences and the carboxyl domain interacts with the coretype sequencess. As a first step to understand the molecular mechanism of the integrase-DNA interaction at the arm-type site, the int(am)94 gene carrying an amber mutation at the 94th codon of the int was cloned under the control of the P$\_$tac/ promoter and the lacI$\_$q/ gene. The Int(am)94 mutant protein of amino terminal 93 amino acid residues can be produced at high level from a suppressor free strain harboring the plasmid pInt(am)94. The arm-type binding activity of Int(am)94 were measured in vivo and in vitro. A comparison of the arm-type binding properties of the wild-type integrase and the truncated Int(am)94 mutant indicated that the truncated fragment containing 93 amino acid residues carry all the determinants for DNA binding at the arm-type sites.