• Journal of Periodontal and Implant Science
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• 제32권2호
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• pp.351-360
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• 2002

In the study presented here, identification, purification, and partial characterization of a hemin-regulated protein in Prevotella nigrescens were carried out. The results of this study confirm that the availability of hemin influences the expression of a selected membrane protein as well as the growth rate of P. nigrescens ATCC 33563. The 50 kDa cell envelope associated protein, whose expression is hemin regulated, is considered to be a putative hemin-binding protein from P. nigrescens. Disulfide bonds were not present in this protein, and N'-terminal amino acid sequence analysis revealed that this protein belongs to a new, so far undescribed protein. The 50 kDa protein was found to be rich in hydrophilic amino acids, with glycine comprising approximately 60% of the total amino acids. The study described here is the first to identify, purify, and biochemically characterize a putative hemin-binding protein from P. nigrescens. Work is in progress to further characterize the molecular structure of this protein.

• Journal of Periodontal and Implant Science
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• 제36권1호
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• pp.155-165
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• 2006

The results of this study confirm that the availability of hemin influences the expression of selected membrane proteins of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens. A 30 kDa (heated 24 kDa) hemin-binding protein whose expression is hemin regulated was identified and purified in P. gingivalis. A strong hemin-binding function was found by LDS-PAGE and TMBZ staining when P. gingivalis cells were grown under hemin-limited conditions. A 50 kDa cell envelope associated protein, whose expression is hemin regulated, is considered to be a putative hemin binding protein from P. intermedia and P. nigrescens, respectively. N-terminal amino acid sequence analysis of CNBr-digested 24 kDa hemin binding protein from P. gingivalis revealed that this protein belongs to a new, so far undescribed hemin-binding class of proteins. N-terminal amino acid sequence of a 50 kDa putative hemin binding protein from P. intermedia was identical with Enolase from Streptococcus intermedia. Work is in progress to further characterize the molecular structure of these proteins.

• Journal of Microbiology and Biotechnology
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• 제22권4호
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• pp.470-478
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• 2012

Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane ${\beta}$-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.

• 대한바이러스학회지
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• 제27권2호
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• pp.209-216
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• 1997

Three dimensional structures of envelope protein from Korean isolates and Nakayama-NIH strain of Japanese encephalitis virus (JEV) were deduced by a computer program (HyperChem 4.0 Chemplus 1.0) based on the data of the three dimentional structure of Tick-borne encephalitis virus. In the three dimensional structure of envelope protein, neutralizing epitope and T-helper cell recognition site of C-terminal region of Korean isolates were structually similar to those of Nakayama-NIH but the N-terminal region was not. Korean JE isolates were compared with Nakayama-NIH strain by using cross-neutralization antibody test. Neutralizing activities of Korean isolates derived from guinea pigs were higher than those of Nakayama-NIH strain against Korean isolates, although the polyclonal antibody titers of Nakayama-NlH showed 1:160 to 1:640 against Korean isolates. According to the results from three dimentional structures and cross-neutralization analyses, the antigenic difference between Korean JE isolates and Nakayama-NIH strain may be dependent on structural difference of envelope protein.

• 대한바이러스학회지
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• 제27권1호
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• pp.9-17
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• 1997

Hepatitis C virus (HCV) E2 protein is known to be one of putative envelope proteins. To develop a sensitive detection method for HCV infected tissues and cells, monoclonal antibodys (MAbs) to the E2 protein of HCV were prepared from mice immunized with recombinant baculovirus-expressing E2 protein (Bac-E2). Several hybridoma clones secreting various levels of MAb were isolated and isotypes of these MAb were determined. One clone (L.2.3.3) was used for ascites production and the E2-MAb was purified and characterized. The L.2.3.3 reacted well with both Bac-E2 and E. coli expressed glutathione-S-transferase-E2 (GST-E2) fusion proteins. Using HCV patient sera, E2 envelope protein was found to be localized in the cell membrane boundary both in CHO cells and insect cells which express HCV E2 protein. Similar result was obtained when same cells were treated with the MAb L.2.3.3. These results demonstrated that Bac-E2 protein is capable of eliciting high titer antibody production in mice.

• 대한치주과학회:학술대회논문집
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• 대한치주과학회 2002년도 제42회 종합학술대회 연제초록
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• pp.87-87
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• 2002

• 한국가축번식학회지
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• 제19권4호
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• pp.293-305
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• 1996

Retrovirus는 DNA가 아닌 RNA를 유전 물질로 갖고 있는 동물 virus인데 각 virus는 RNA와 함께 크게 gag, pol. 그리고 env 등의 3가지 단백질로 구성되어 있다. gag 단백질은 virus의 내부구조를 형성하는 단백질이고, pol단백질은 감염을 통해 표적 세포에 도입된 retrovirus의 RNA를 DNA로 역전사시키는 reverse transcriptase의 역할을 하며, env단백질은 virusdml 외부를 구성하는 단백질로써 이 단백질에 의해 각 retrovirus의 종류에 따른 감염이 가능한 표적세포의 종류가 결정된다(host cell specificity). 따라서 어떤 retrovirus의 envelope단백질과 표식세포에 있는 retrovirus의 envelope 단백질에 대한 특정 receptor와의 상호 작용에 의해 세포속으로 도입된 virus의 RNA는 reverse transcriptase에 의해 DNA로 역전사된 후 표적세포의 genomic DNA에 삽입되는 특징을 가진다. 이러한 특징을 가진 retrovirus vector system은 형질 전환 동물의 생산에 있어서 현재까지의 주된 방법인 수정란의 pronucleus에의 DNA microinjection방법 보다 여러 가지 면에서 우수함에도 불구하고 쥐 이외의 다른 동물에서는 거의 이용되고 있지 않는 실정이다. 주된 원인으로는 현재 사용되고 있는 대부분의 retrovirus vector system이 쥐의 백혈병 virus를 근간으로 하기 때문에 이 system에서 생산된 virus는 쥐 이외의 다른 동물, 특히 유제류의 세포에는감염성이 아주 약하기 때문이다. 이러한 결점을 해결하기 위하여 최근에 기존의 쥐 백혈병 virus의 envelope protein을 vesicular stomatitis virus의 G protein으로 대체한 hybrid retrovirus vector system이 개발되었다. 이러한 system에서 생산되는 virus는 조류를 포함한 거의 모든 종류의 동물세포를 감염시킬 수 있으며 몇몇 특정세포에 대해서는 기존의 retrovirus vector system에 비해 1,000배 이상의 높은 감염도를 나타내는데 그 특징이 있다. 따라서 이러한 새로운 virus vector system을 이용할 경우, 보다 다양한 종에 있어서 형질전환 동물을 효율적으로 생산할 수 있을 뿐만 아니라 형질전환 동물의 생산 방법 자체를 다양화 시킬 수 있다고 본다.

• Journal of Microbiology
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• 제45권1호
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• pp.41-47
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• 2007

The Hantaan virus (HTNV) is an enveloped virus that is capable of inducing low pH-dependent cell fusion. We molecularly cloned the viral glycoprotein (GP) and nucleocapsid (NP) cDNA of HTNV and expressed them in Vero E6 cells under the control of a CMV promoter. The viral gene expression was assessed using an indirect immunofluorescence assay and immunoprecipitation. The transfected Vero E6 cells expressing GPs, but not those expressing NP, fused and formed a syncytium following exposure to a low pH. Monoclonal antibodies (MAbs) against envelope GPs inhibited cell fusion, whereas MAbs against NP did not. We also investigated the N-linked glycosylation of HTNV GPs and its role in cell fusion. The envelope GPs of HTNV are modified by N-linked glycosylation at five sites: four sites on G1 (N134, N235, N347, and N399) and one site on G2 (N928). Site-directed mutagenesis was used to construct eight GP gene mutants, including five single N-glycosylation site mutants and three double-site mutants, which were then expressed in Vero E6 cells. The oligosaccharide chain on residue N928 of G2 was found to be crucial for cell fusion after exposure to a low pH. These results suggest that G2 is likely to be the fusion protein of HTNV.

• 대한바이러스학회지
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• 제29권4호
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• pp.221-233
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• 1999

Prospect Hill Virus (PHV) is the well known serotype of hantavirus, a newly established genus in family Bunyaviridae. Extensive studies have upheld the original view of PHV genetics with three genes such as nucleocapsid (N) protein, envelope proteins (G1, G2) and RNA dependent RNA polymerase. In this study, we report the existence of additional gene that is encoded in an overlapping reading frame of the N protein gene within S genome segment of PHV. This gene is expected to encode a nonstructural small (NSs) protein and it seems to be only found in PHV infected cell. The presence and synthesis of NSs protein could be demonstrated in the cell infected with PHV using anti-peptide sera specific to the predicted amino acid sequence deduced from the second open reading frame. Ribosomal synthesis of this protein appears to occur at AUG codon at the 83rd base of S genome segment, downstream of N protein initiation codon. This protein is small in size (10.4 KDa) and highly basic in nature. The expression strategy of NSs protein appears that a signal mRNA is used to translate both N and NSs protein in PHV infected cell. 10 KDa protein in virus infected cell lysates can bind to mimic dsRNA. This fact strongly suggests that NSs protein may be involved in virus replication on late phase of viral life cycle.

• 한국가축번식학회지
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• 제19권1호
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• pp.35-41
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• 1995

본 연구는 형질전화우의 생산성 제고를 위한 일환으로서 새로운 기법인 retrovirus vector system의 이용성을 검토하고자 실시하였다. Retrovirus-producing cell은 미세주입법을 이용하여 체외생산된 1.5일(1~4-세포기) 수정란의 위란강에 주입(5~10 cells/embryo) 되었으며, 이때 사용된 retrovirus-producing cell line은 Gibbon ape leukemia virus (GaLV) envelope protein에 encapsidation되어 replication-defective retrovirus를 분비하도록 제작되었다. 주입된 유전자의 표지유전자로서 E. coli LacZ 유전자를 사용하였으며, X-gal 염색법은 발달이 유도된 상실배와 배반포 단계에 실시하여 LacZ 유전자의 발현 유무를 확인하였다. 이 실험의 결과를 요약하면 다음과 같다. 1. Virus의 infectivity를 높이기 위해 사용된 polybrene의 최저농도는 5$\mu\textrm{g}$/ml이었다. 2. Retrovirus-producing cell이 주입된 1.5일 수정란의 상실배기와 배반포기로의 발달율은 29%였다. 3. 이 때의 LacZ+ 발현율은 21%였다. 4. 본 실험에 사용된 retrovirus-producing cell은 replication-competent retroviruses를 생산해 내지않는다는 것을 확인할 수 있었다.