• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.552-559
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• 2003

The FimH subunit of type 1-fimbriated Escherichiu coli (E. coli) has been determined as a major cause for urinary tract infections. Thus, to produce a possible vaccine antigen against urinary tract infections, the fimIH gene was genetically coupled to the ctxa2b gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimH/ctxa2b was then transformed into E. coli K-12 TB1 and its nucleotide sequence was verified. A fusion protein, based on fusing adhesin to the cholera toxin subunit A2B (CTXA2B), was induced with 0.01 mM isopropyl-${\beta}-D-thiogalactoside$ (IPTG) for 4 h at $37^{\circ}C$ to yield a soluble fusion protein. The fusion protein was then purified by affinity chromatography. The expressed fusion protein was confirmed by SDS-PAGE and Western blotting using antibodies to the maltose binding protein (MBP) or the cholera toxin subunit B (CTXB), plus the N-terminal amino acid sequence was also analyzed. The orderly-assembled fusion protein was confirmed by a modified $G_{Ml}-ganglioside$ ELISA, using antibodies to adhesin. The results indicated that the purified fusion protein was an adhesin/CTXA2B protein containing E. coli adhesin and the $G_{Ml}-ganglioside$ binding activity of CTXB. Accordingly, this adhesin/CTXA2B protein may be a potential antigen for oral immunization against uropathogenic E. coli.

• Molecules and Cells
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• 제19권1호
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• pp.54-59
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• 2005

Deoxyribonuclease I (DNase I) is a divalent cation dependent endonuclease and thought to be a significant barrier to effective gene delivery. The only known DNase I-specific inhibitor is monomeric actin which acts by forming a 1:1 complex with DNase I. Its use, however, is restricted because of tendency to polymerize under certain conditions. We screened two random phage peptide libraries of complexity $10^8$ and $10^9$ for DNase I binders as candidates for DNase I inhibitors. A number of DNase I-binding peptide sequences were identified. When these peptides were expressed as fusion proteins with Escherichia coli maltose binding protein, they inhibited the actin-DNase I interaction ($IC_{50}=0.1-0.7{\mu}M$) and DNA degradation by DNase I ($IC_{50}=0.8-8{\mu}M$). Plasmid protection activity in the presence of DNase I was also observed with the fusion proteins. These peptides have the potential to be a useful adjuvant for gene therapy using naked DNA.

• Biomolecules & Therapeutics
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• 제13권2호
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• pp.101-106
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• 2005

The generation of secretory IgA antibodies (Abs) for specific immune protection of mucosal surfaces depends on stimulation of the mucosal immune system, but this is not effectively achieved by parenteral or even oral administration of most soluble antigens. Thus, to produce a possible vaccine antigen against urinary tract infections, the uropathogenic E. coli (UPEC) adhesin was genetically coupled to the heat-labile Escherichia coli enterotoxin A2B (ltxa2b) gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimH/ltxa2b was then transformed into E. coli K-12 TB1 and its nucleotide sequence was verified. The chimeric protein was then purified by applying the affinity chromatography. The purified chimeric protein was confirmed by SDS-PAGE and westem blotting using antibodies to the maltose binding protein (MBP) or the heat labile E. coli subunit B (LTXB), plus the N-terminal amino acid sequence was analyzedd. The orderly-assembled chimeric protein was confirmed by a modified $G_{M1}$-ganglioside ELISA using antibodies to adhesin. The results indicate that the purified chimeric protein was an Adhesin/LTXA2B protein containing UPEC adhesin and the $G_{M1}$-ganglioside binding activity of LTXB. thisstudy also demonstrate that peroral administration of this chimeric immunogen in mice elicited high level of secretory IgA (sIgA) and serum IgG Abs to the UPEC adhesin. The results suggest that the genetically linked LTXA2B acts as a useful mucosal adjuvant, and that adhesin/LTXA2A chimeric protein might be a potential antigen for oral immunization against UPEC.

• Biomolecules & Therapeutics
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• 제11권3호
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• pp.157-162
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• 2003

The generation of secretory IgA antibodies(Abs) for specific immune protection of mucosal surfaces depends on stimulation of the mucosal immune system, but this is not effectively achieved by parenteral or even oral administration of most soluble antigens. Thus, to produce a possible vaccine antigen against urinary tract infections, the uropathogenic E. coli (UPEC) adhesin was genetically coupled to the ctxa2b gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimHIctxa2b was then transformed into E. coli K-12 TB1 and its nucleotide sequence was verified. The chimeric protein was then purified by applying the affinity chromatography. The purified chimeric protein was confirmed by SDS-PAGE and western blotting using antibodies to the maltose binding protein (MBP) or the cholera toxin subunit B (CTXB), plus the N-terminal amino acid sequence was analyzed. The orderly-assembled chimeric protein was confirmed by a modified $G_{M1}$-ganglioside ELISA using antibodies to adhesin. The results indicate that the purified chimeric protein was an Adhesin/CTXA2B protein containing UPEC adhesin and the $G_{M1}$-ganglioside binding activity of CTXB. This study also demonstrate that peroral administration of this chimeric immunogen in mice elicited high level of secretory IgA and serum IgG Abs to the UPEC adhesin. The results suggest that the genetically linked CTXA2B acts as a useful mucosal adjuvant, and that the adhesin/CTXA2B chimeric protein might be a potential antigen for oral immunization against UPEC.

• BMB Reports
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• 제43권5호
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• pp.319-324
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• 2010

Experimental bioinformatics data obtained from an E. coli cell-based eukaryotic protein purification experiment were analyzed in order to identify any bottleneck as well as the factors affecting the target purification. All targets were expressed as His-tagged maltose-binding protein (MBP) fusion constructs and were initially purified by immobilized metal affinity chromatography (IMAC). The targets were subsequently separated from the His-tagged MBP through TEV protease cleavage followed by a second IMAC isolation. Of the 743 total purification trials, 342 yielded more than 3 mg of target proteins for structural studies. The major reason for failure of target purification was poor TEV proteolysis. The overall success rate for target purification decreased linearly as cysteine content or isoelectric point (pI) of the target increased. This pattern of pI versus overall success rate strongly suggests that pI should be incorporated into target scoring criteria with a threshold value.

• 한국미생물·생명공학회지
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• 제21권3호
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• pp.229-238
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• 1993

An nlp (Ner like protein) gene from E. coli was previously cloned and sequenced. Here we show that expression of the sugar metabolism related genes, lacZ, malQ and malP, increased 2.5-to 8.3-fold in the presence of a plasmid containing the nlp gene. This suggested that the nlp gene could induce maltose- and lactose-metabolism coordinately with crp*1 in the absence of cAMP. Using the nlp-lacZ fusion gene, it was possible to show the promoter of nlp was active in vivo.

• BMB Reports
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• 제33권6호
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• pp.440-447
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• 2000

In this study HepG2 cells were used to express and purify HBV pol proteins. In order to facilitate purification of HBV pol proteins, HBV pol and its deletion mutants were fused to MBP (Maltose Binding Protein). As a result we successfully expressed and partially purified both wild type and mutant recombinant HBV pol proteins by using an amylose resin and anti-MBP antibody. In the case of wild type, the anti-MBP antibody detected three bands. One was full-length and the others were generated by proteolysis of the terminal domain region. The expressed MBP/POL proteins were localized both in the cytoplasm and in the perinuclear region. The purified proteins had polymerase activity toward an exogenous homo-polymer template. The MBP/POL protein also had DNA synthesis activity in vivo, since the MBP/POL expression construct was able to complement a HBV polymerase mutant in trans.

• 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.

• 수산해양교육연구
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• 제27권5호
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• pp.1470-1478
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• 2015

Adjuvant is an immune enhancer commonly used during vaccination to enhance the host immune response. In the present study, we produced the several recombinant protein from immune related gene of olive flounder (Paralichthys olivaceus). Especially, to produce the soluble type of recombinant protein, we constructed the MBP (Maltose binding protein) fusion G-CSF (Granulocyte colony stimulating factor) recombinant protein among the flounder immune related genes. To verify the immune stimulatory effect and safety of this recombinant protein (rPoGCSF), expression changes of several immune genes were tested using quantitative real-time PCR method with gene specific primer from flounder head kidney leukocytes. As a result, we confirmed that the rPoGCSF has an ability of immune stimulatory effect, also it has broad range of pH and temperature.

• Journal of Microbiology and Biotechnology
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• 제8권1호
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• pp.34-41
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• 1998

A method was developed for the controlled expression of an antimicrobial peptide magainin in Escherichia coli. A series of concatemeric magainin genes was constructed with a gene amplification vector, and fused to the 3'end of malE gene encoding the affinity ligand, E. coli maltose-binding protein (MBP). The construct directed the synthesis of the fusion protein with the magainin polypeptide fused to the C-terminus of MBP. The fusion protein was expressed in a tightly regulatable expression system which was under the control of an invertible promoter. The MBP-fused magainin monomer was expressed efficiently. However, the expression level of the MBP-fused magainin in E. coli decreased with the increasing size of multimers possibly because of the transcription and translation inhibition by the multimeric peptides. After purification using an amylose affinity column, the fusion protein was digested by factor Xa at a specific cleavage site between the monomers. The recombinant magainin had an antimicrobial activity identical to that of synthetic magainin. This experiment shows that a biologically active, antimicrobial peptide magainin can be produced by fusing to MBP, along with a promoter inversion vector system.