• Biotechnology and Bioprocess Engineering:BBE
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• 제3권2호
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• pp.82-86
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• 1998

The maltose binding protein (MBP) fusion protein system is a versatile tool to express and isolate recombinant proteins in E. coli. In this system, MBP fusion proteins are efficiently isolated from whole cell lysate using amylose conjugated agarose beads and then eluted by competition with free maltose. Since MBP is a rather large molecule (∼42 kDa), for further experiments, the MBP part is usually proteolytically cleaved from the fusion protein and subsequently removed by ion-exchange chromatography or rebinding to amylose columns after washing out excess and MBP-bound maltose. In the present study, we have developed an improved method for the removal of cleaved MBP, which is advantageous over conventional methods. In this method, factor Xa cleaved MBP fusion proteins were incubated with Sepharose beads conjugated with MBP specific monoclonal antibodies and then precipitated buy centrifugation, resulting in highly purified proteins in the supernatant.

• Parasites, Hosts and Diseases
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• 제54권4호
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• pp.461-469
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• 2016

Giardia lamblia is a protozoan that causes diarrheal diseases in humans. Cytoskeletal structures of Giardia trophozoites must be finely reorganized during cell division. To identify Giardia proteins which interact with microtubules (MTs), Giardia lysates were incubated with in vitro-polymerized MTs and then precipitated by ultracentifugation. A hypothetical protein (GL50803_8405) was identified in the precipitated fraction with polymerized MTs and was named GlMBP1 (G. lamblia microtubule-binding protein 1). Interaction of GlMBP1 with MTs was confirmed by MT binding assays using recombinant GlMBP1 (rGlMBP1). In vivo expression of GlMBP1 was shown by a real-time PCR and western blot analysis using anti-rGlMBP1 antibodies. Transgenic G. lamblia trophozoites were constructed by integrating a chimeric gene encoding hemagglutinin (HA)-tagged GlMBP1 into a Giardia chromosome. Immunofluorescence assays of this transgenic G. lamblia, using anti-HA antibodies, revealed that GlMBP1 mainly localized at the basal bodies, axonemes, and median bodies of G. lamblia trophozoites. This result indicates that GlMBP1 is a component of the G. lamblia cytoskeleton.

• 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.

• Toxicological Research
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• 제16권3호
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• pp.221-227
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• 2000

A 6-sulfooxymethylbenzo[a]pyrene (SMBP), the ultimate metabolite of methyl-substituted benzo[a]pyrene (BP), has been found to be carcinogenic in mice. These properties may be attributable to its strong reactivity with cellular macromolecules such as DNA. However, serum and its major constituent albumin attenuated significantly the cytotoxicity and mutagenicity of 5MBP in bacterial and mammalian cell systems. This inhibitory activity of serum against 5MBP-induced cytotoxicity and mutagenicity in Chinese hamster V79 cells appears to be caused by the reduced macromolecular adducts such as DNA and proteins, but serum failed to reduce 5MBP binding to naked calf thymus DNA. A number of proteins in the serum could act as nucleophiles that are able to intercept reactive chemicals through covalent binding. Albumin present in the plasma seems to be one of major components responsible for direct binding with 5MBp, thereby reducing its reactivity to genetic materials. We here determined which fraction is preferential for 5MBP binding through fractionation of 5MBP-treated serum with ammonium sulfate. The albumin-containing fraction had slightly more affinity for 5MBP than the immunoglobulin-containing fraction. Our results indicate that the covalent modification of plasma proteins may reduce 5MBP-induced damage.

• 대한화학회지
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• 제41권4호
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• pp.205-209
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• 1997

신경수초 염기성 단백질(MBP)에 대한 탈인산화의 자리 특이성에 대한 연구를 시험관에서 수행하였다. MBP에서 인산화된 자리를 알아내기 위해서 MBP를 단백질 키나제 C로 인산화 시키고 단백질 탈인산화 효소 PP2A로 탈인산화시켰다. 인산화된 MBP는 트립신으로 잘라내어서 역상 HPLC 크로마토그래피로 펩티드 조각들을 분리하였다. 각 조각들을 섬광 계측한 후 일부 펩티드의 아미노산 서열을 결정하였다. 일곱 개의 방사능을 보이는 펩티드 조각이 검출되었으며 두번째 조각($P_2$)의 $Ser^{55}$에 해당하는 아미노산이 인산화 반응에 가장 큰 민감도를 보였다. 그러나 탈인산화는 다섯번째 고작($P_5$)의 인산이 가장 잘 방출되는 것으로 나타났다. 이 결과는 MBP가 단백질 탈인산화 반응에 적절한 기질임을 보여주고 있다.

• Journal of Microbiology and Biotechnology
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• 제25권2호
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• pp.274-279
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• 2015

Receptor activator of nuclear factor-kappa B ligand (RANKL) is a critical factor in osteoclastogenesis. It makes osteoclasts differentiate and multinucleate in bone remodeling. In the present study, RANKL was expressed as a soluble maltose binding protein (MBP)-fusion protein using the Escherichia coli maltose binding domain tag system (pMAL) expression vector system. The host cell E. coli DH5α was cultured and induced by isopropyl β-_D-1-thiogalactopyranoside for rRANKL expression. Cells were disrupted by sonication to collect soluble MBP-fused rRANKL. The MBP-fusion rRANKL was purified with MBP Trap affinity chromatography and treated with Tobacco Etch Virus nuclear inclusion endopeptidase (TEV protease) to remove the MBP fusion protein. Dialysis was then carried out to remove binding maltose from the cleaved rRANKL solution. The cleaved rRANKL was purified with a second MBP Trap affinity chromatography to separate unsevered MBP-fusion rRANKL and cleaved MBP fusion protein. The purified rRANKL was shown to have biological activity by performing in vitro cell tests. In conclusion, biologically active rRANKL was successfully purified by a simple two-step chromatography purification process with one column.

• 생명과학회지
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• 제25권4호
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• pp.385-389
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• 2015

선행연구에서 카스파제-3 효소가 DEVD 기질을 완전히 절단하는 반면 IETD 기질은 DEVD의 약 50%정도만을 부분적으로 분해한다는 사실을 보고하였다. 본 연구에서는 정제된 폴리-단백질이 카스파제-3 단백질 분해효소의 기질에 따른 차별적인 분해활성에 의해 프로세싱 되는 양상을 분석하였다. 모델 단백질로서 GST, MBP, RFP 세 종류의 단백질을 DEVD 및 IETD 펩타이드로 연결시킨 폴리-단백질을 제작하였으며, 폴리-단백질은C-말단에 6개의 히스티딘 태그가 결합되도록 클로닝 되었다. IMAC 크로마토그래피를 이용하여 분리 및 정제된 재조합 단백질은 SDS-PAGE 분석을 통해 분자량이 약 93 kDa으로 나타났으며, 카스파제-3 효소의 처리에 의해 각각 MBP:RFP, MBP 그리고 GST 3종류의 단백질 절편으로 절단 및 분리되었다. 이 연구를 통해 단백질 분해효소와 기질 간의 반응성의 차이를 이용하여 폴리-단백질을 정량적으로 프로세싱 할 수 있다는 예를 보여주었다.

• 산업기술연구
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• 제28권B호
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• pp.59-63
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• 2008

Rapid production of therapeutic proteins such as angiostatin and endostatin angiogenic inhibititors has been highly demanded for cancer treatment. In this regard, recombinant human angiostatin and endostatin were successfully expressed as soluble forms by maltose binding protein (MBP)-mediated fusion expression in Escherichia coli. PCR amplified, angiostatin and endostatin genes from human placenta cDNA library were inserted into an expression vector pMAL-c2e to construct prokaryotic expression vectors, pMAL-c2e/AS and pMAL-c2e/ES, respectively. Recombinant angiostatin and endostatin were efficiently expressed in E. coli origami (DE3) after IPTG induction and protein expression were confirmed by SDS-PAGE analyses. The expressed recombinant proteins were purified near homogenity using an amylose affinty column chromatography. In contrast that previous E. coli expressions were all insoluble, our results first time demonstrated that MBP fused human angiostatin and endostatin were soluble in E. coli.

• The Korean Journal of Ecology
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• 제21권3호
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• pp.225-231
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• 1998

The studies on the potentiality of biomonitoring heavy metal pollution in coastal region of industrial complex were performed to investigate the heavy metal accumulation and induction of metal-binding protein (MBP) as detoxification process using Rumex maritimus. Bioconcentration in organs and MBP in root of R. maritimus was investigated for the research of the tolerance of heavy metals. The bioconcentration of cadmium and zinc in organs showed 3.6-8.0 times in root higher than in shoot, so it was found that heavy metal accumulated selectively in root. MBP increased absorbance in 254 nm and decreased in 280 nm, because it was composed of high cystein content and low aromatic acids, so absorbance had large difference between 254 nm and 280 nm. The existence of MBP in the 10-20 fraction was ascertained with anion exchange chromatography and it was identified that concentration of heavy metal increased according as an exposure concentration of medium increased in QAE Sephadex A-25 elution profile. These results suggested that MBP could play a role in biomarker determining the bioconcentration of plant. This study demonstrated a possibility that removal ability of heavy metal of R. maritimus resulted from detoxification process and MBP could be utilized as a biomarker of heavy metal pollution.

• 화약ㆍ발파
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• 제22권3호
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• pp.79-84
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• 2004

일반적으로 유중수형 에멀젼폭약은 제조공정이나 사용조건에서 안전하다고 알려져 있다. 그러나 1975년 캐나다에서 벌크 에멀젼폭약을 펌프로 이송 중에 발생한 폭발사고와 같이 원치 않는 사고가 발생할 가능성이 있다. 에멀젼폭약은 어떠한 조건하에서 착화하는 경우 연소에서 폭굉으로 전이(Deflagration-to-Detonation Transition, 약칭 DDT)하는 현상이 발생하며 또한 연소가 온도에 따라 지속되는 최소 압력(Minimum Burning Pressure, 약칭 MBP)이 존재하며 최소 압력이하로 유지할 경우 착화되어도 바로 연소가 중단되는 것으로 알려져 있다. 본 연구는 에멀젼폭약 제조공정의 안전성 평가를 목적으로 다양한 에멀젼폭약을 제조, 밀폐용기 내에서 최소연소압력(MBP) 측정 시험을 실시하였다. 본 실험에서 수분함량 6%에서 20%, 알루미늄 함량 1%에서 11%까지 함유한 시료를 각각 제조하여 밀폐용기 내에서 열원의 온도별로 실시하였다. 실험결과 수분함량 6% 시료의 최소연소압력(MBP)이 가장 낮은 3 bar를 나타냈으며 수분함량 18%이상에서는 100bar이상에서도 연소가 지속되지 않았다. 알루미늄 함량에 따라서는 큰 변화는 보이지 않았으나 알루미늄을 함유하지 않는 것에 비해 최소연소압력(MBP)가 비교적 높게 나타났다. 따라서 향후 에멀젼폭약 제조공정의 안전성 향상을 위하여 제조하는 에멀젼폭약의 최소연소압력(MBP)를 측정, 공정압력조건을 MBP 이하로 관리할 때 안전을 보증할 수 있을 것으로 판단된다.