• BMB Reports
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• 제41권8호
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• pp.568-574
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• 2008

Antisense RNA molecules are powerful tools for controlling the expression of specific genes but their use in prokaryotes has been limited by their unpredictable antisense effectiveness. Moreover, appreciation of the molecular mechanisms associated with silencing in bacteria is still restricted. Here we report our attempts to define an effective antisense strategy in E. coli, and to dissect the observed silencing process. Antisense constructs complementary to different regions of lacZ were investigated, and silencing was observed exclusively upon expression of antisense RNA hybridising the 5'UTR of lac messenger. The level of lacZ mRNA was reduced upon expression of this antisense construct, and the silencing competence was found to be closely associated with its stability. These observations may help in the design of antisense molecules directed against prokaryotic genes.

• Journal of Microbiology and Biotechnology
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• 제12권2호
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• pp.306-311
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• 2002

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslationally modified compound in eukaryotic cells. Human O-GlcNAc transferase (OGT) was produced as a maltose binding protein (MBP) fusion protein, which showed significant catalytic activity to modify recombinant Sp1, transcription factor. To facilitate the production of O-GlcNAc modified proteins, instead of using the tedious in vitro glycosylation reaction or expression in eukaryotic cells, a MBP-fusion OGT expression vector (pACYC184-MBPOGT) was constructed using pACYC184 plasmid, which could coexist with general prokaryotic expression vectors containing ColE1 origin. By cotransforming pACYC184-MBPOGT and pGEX-2T vectors into Escherichia coli BL21, intracellular O- GlcNAcylated proteins could be obtained by a simple purification procedure. It is expected that this may be a useful tool for production of O-GlcNAc modified proteins.

• 대한수의학회지
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• 제45권2호
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• pp.185-189
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• 2005

Actinobacillus pleuropneumoniae is the causative agent of a porcine contagious pleuropneumonia. Among several virulence factors including exotoxin (Apx toxins), LPS, transferrin-binding proteins, OMPs, and some proteases, Apx toxins have been major targets for the protection study. In this study, cloning and expression of A. pleuropneumoniae Apx I and Apx II toxin, which are produced by all highly virulent strains, were performed by Escherichia coli expression system. Genes coding Apx I and II toxin were amplified from the A. pleuropneumoniae serotype 5 genomic DNA using polymerase chain reaction and cloned to a prokaryotic expression vector, pRSET. Expression of the Apx I and Apx II coding sequences in E. coli resulted in the formation of insoluble inclusion bodies purified according to a denaturing purification protocol, which employs the use of guanidium. Recombinant proteins were purified using $Ni^{2+}$-charged resin affinity purification. This expression and purification system made it possible to produce Apx I and Apx II in large amounts for further immunologic studies.

• International Journal of Industrial Entomology and Biomaterials
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• 제17권2호
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• pp.189-195
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• 2008

The sequence of hydroxypyruvate isomerase gene was obtained in NCBI. In this study, the hydroxypyruvate isomerase gene of Bombyx.mori was identified and annotated with bioinformatics tools. The result was confirmed by RT-PCR, prokaryotic expression, mass spectrographic analysis and sub-cellular localization. The hydroxypyruvate isomerase cDNA comtains a 783bp ORF, and has 4 exons. The deduced protein has 260 amino acid residues with the predicted molecular weight of 29169.30 Da, isoelectric point of 6.10, and contains conserved PRK09997 and Hfi domains. The hydroxypyruvate isomerases of Nasonia vitripennis and Bombyx mori have a high homology. Through RTPCR analysis, we found that this transcript was present in testis, ovary, blood-lymph, fat body, midgut, silk gland and tuba Malpighii. This protein was located in cytoplasm through immunohistochemistry. We submitted the cloned gene under the accession number EU344910. The enzyme has been classified under accession number EC 5.3.1.22.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권3호
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• pp.159-163
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• 2000

The genome sequencing project has generated and will contitute to generate enormous amounts of sequence data. Since the first complete genome sequence of bacterium Haemophilus in fluenzae was published in 1995, the complete genome sequences of 2 eukaryotic and about 22 prokaryotic organisms have detemined. Given this everincreasing amounts of sequence information, new strategies are necessary to efficiently pursue the phase of the geome project- the elucidation of gene expression patterns and gene product function on a whole genome scale. In order to assign functional information to the genome sequence, DNA chip technology was developed to efficienfly identify the differential expression pattern of indepondent biogical samples. DNA chip provides a new tool for genome expreesion analysis that may revolutionize revolutionize many aspects of human kife including mew surg discovery and human disease diagnostics.

• Journal of Microbiology and Biotechnology
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• 제20권6호
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• pp.974-977
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• 2010

Cyclophilins contain the conserved activity of cis-trans peptidyl-prolyl isomerase, which is implicated in protein folding, and function as molecular chaperones. When the yeast cyclophilin A gene (cpr1) was subcloned into the prokaryotic expression vector pKM260, it was found that the expression of Cpr1 drastically increased the cell viability of E. coli BL21 when under abiotic stress conditions, as in the presence of cadmium, copper, hydrogen peroxide, heat, and SDS. Therefore, this study illustrates the importance of Cpr1 as a molecular chaperone that can improve the cellular stress responses when E. coli cells are exposed to adverse conditions, while also demonstrating its potential to increase the stability of E. coli strains utilized for the production of recombinant proteins.

• Animal cells and systems
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• 제12권3호
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• pp.149-155
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• 2008

We have isolated and characterized Agrius convolvuli cDNA encoding a c-type lysozyme. The cDNA sequence encodes a processed protein of 139 amino acid residues with 19 amino acid residues amino-terminal signal sequence and 120 amino acid residues mature sequence. The amino acid residues responsible for the catalytic activity and the binding of the substrate are conserved. Agrius lysozyme has a high identity to Manduca sexta. Recombinant A. convolvuli lysozyme was expressed in Escherichia coli BL21(DE3) pLysS cells for pGEX 4T-1 expression vector. Their optimal conditions for the fusion protein expression and purification were screened. Lysozyme gene amplified with primers ACLyz BamHI and ACLyz XhoI was ligated into the pGEX 4T-1 vector, which contained the glutathione S-transferase(GST) gene for fusion partner. The fusion protein was induced by IPTG and identified by SDS-PAGE analysis. Molecular weight of the fusion protein was estimated to be about 45 kDa. Recombinant lysozyme, fused to GST, was purified by glutathion-Sepharose 4B affinity chromatography. Western blot analysis of this protein revealed an immunoreactivity with the anti-Agrius lysozyme.

• 대한수의학회지
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• 제46권1호
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• pp.13-19
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• 2006

Generally known psittacosis or ornithosis is a disease of birds caused by the bacterium Chlamydia psittaci. Humans are accidential hosts and are most commonly infected from avian sources. It raises hepatitis or neurosis. As major outer membrane protein (MOMP) of Chlamydia psittaci has been known to play a role in the avoidance of host immune defenses, research on developing a Chlamydia vaccine has focused on the MOMP. In this study, the gene encoding the major outer membrane protein (MOMP) of the Chlamydia psittaci strain 6BC was cloned and expressed in Escherichia coli strain M-15. The recombinant DNA was cloned by fusion prokaryotic expression vector pQE30-GFPII. Expression of the recombinant protein was performed in E. coli and was induced by IPTG. The size of expressed recombinant protein is 74.220 kDa (MOMP, 43.260 kDa; GFP expression region, 30 kDa; $6{\times}His$ tag, 960Da). This protein was purified by using his-tagging-inclusion body. Recombinant protein was reconfirmed through ELISA test and western blot with antibody against pQE30-GFPII. It will be useful antibody development.

• 대한바이러스학회지
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• 제28권1호
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• pp.21-30
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• 1998

Synthetic genes encoding the gag p24 and the part of the envelope protein gp41 of the human immunodeficiency virus (HIV-1) were cloned and overexpressed as fusion proteins in Escherichia coli, using an expression vector carrying T7 promoter and the poly-histidine leader, sequence. The overexpressed p24 fusion protein was purified by centrifugation, Ni-affinity chromatography and CM-sepharose chromatography. The overexpressed gp41 fusion protein was purified by centrifugation, $C_4$ chromatography and DEAE-sepharose chromatography. The purified fusion proteins showed a high level of purity and immunoreactivity in SDS-polyacrylamide gel electrophoresis and western blot analysis. These results suggest that this prokaryotic - expression purification method is suitable for obtaining a large amount of the viral antigen which may be useful for screening of antibodies to HIV-1 in human blood samples.

• 미생물학회지
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• 제54권1호
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• pp.1-8
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• 2018

원핵 세포는 핵양체 결합 단백질(NAP)로 알려진 다양한 히스톤 유사 단백질을 가지고 있다. 이들은 DNA의 AT-rich 서열에 결합하여, DNA 자체를 감싸거나, 구부리거나, 떨어져 있는 DNA 가닥을 연결시키는 다리 역할을 하여, 결국에는 원핵 생물의 유전자 발현을 조절한다. NAP는 특히 전사의 억제 기능을 가지고 있기 때문에, 유전자 발현 억제에 있어서 이들의 역할과, 구체적인 메커니즘을 밝히는 것을 매우 중요한 일이다. 본 논문에서는 잘 알려져 있는 NAP인 H-NS와 HU에 대하여 정리하였고, 특히 E. coli와 Salmonella Typhimurium에서 이들의 유전자 발현에 대한 기능을 요약하였다. H-NS는 이들의 올리고머화와 필라멘트 구조 형성을 통하여 Salmonella와 같은 사람에 감염하는 병원성 세균의 독성유전자 발현을 억제할 수 있고, 이런 기능을 수행하였을 때 다른 NAP와 함께 작용할 수 있다. 최근에 H-NS는 사람에게 typhoid fever와 systemic disease를 발생시키는 독성물질인, typhoid toxin의 발현 또한 조절할 수 있음이 밝혀졌다. Salmonella에서 HU 또한 독성 유전자뿐만 아니라, 이들의 생리적 기능에 중요한 유전자들의 발현을 조절할 수 있다. 따라서, H-NS와 HU와 같은 NAP들이 원핵 생물의 독성 유전자 발현의 분자적인 메커니즘을 밝히는데 중요한 요소임을 제시한다.