• Journal of Microbiology and Biotechnology
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• 제28권5호
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• pp.776-783
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• 2018

The agarase gene gaa16a was identified from a draft genome sequence of Gilvimarinus agarilyticus JEA5, an agar-utilizing marine bacterium. Recently, three agarase-producing bacteria, G. chinensis, G. polysaccharolyticus, and G. agarilyticus, in the genus Gilvimarinus were reported. However, there have been no reports of the molecular characteristics and biochemical properties of these agarases. In this study, we analyzed the molecular characteristics and biochemical properties of agarases in Gilvimarinus. Gaa16A comprised a 1,323-bp open reading frame encoding 441 amino acids. The predicted molecular mass and isoelectric point were 49 kDa and 4.9, respectively. The amino acid sequence of Gaa16A showed features typical of glycosyl hydrolase family 16 (GH16) ${\beta}$-agarases, including a GH16 domain, carbohydrate-binding region (RICIN domain), and signal peptide. Recombinant Gaa16A (excluding the signal peptide and carbohydrate-binding region, rGaa16A) was expressed as a fused protein with maltose-binding protein at its N-terminus in Escherichia coli. rGaa16A had maximum activity at $55^{\circ}C$ and pH 7.0 and 103 U/mg of specific activity in the presence of 2.5 mM $CaCl_2$. The enzyme hydrolyzed agarose to yield neoagarotetraose as the main product. This enzyme may be useful for industrial production of functional neoagaro-oligosaccharides.

• Molecules and Cells
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• 제46권12호
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• pp.764-777
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• 2023

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli, achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC₅₀ values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.

• Journal of Microbiology and Biotechnology
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• 제22권9호
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• pp.1271-1278
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• 2012

Genetic fusion of two proteins frequently induces beneficial effects to the proteins, such as increased solubility, besides the combination of two protein functions. Here, we study the effects of the bacterial surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a on the folding of a C-terminally fused enhanced green fluorescent protein (EGFP) moiety. Although GFPs are generally unable to adopt a functional confirmation in the bacterial periplasm of Escherichia coli cells, we observed periplasmic fluorescence from a chimera of a 150-amino-acid N-terminal truncation of SgsE and EGFP. Based on this finding, unfolding and refolding kinetics of different S-layer-EGFP chimeras, a maltose binding protein-EGFP chimera, and sole EGFP were monitored using green fluorescence as indicator for the folded protein state. Calculated apparent rate constants for unfolding and refolding indicated different folding pathways for EGFP depending on the fusion partner used, and a clearly stabilizing effect was observed for the SgsE_C fusion moiety. Thermal stability, as determined by differential scanning calorimetry, and unfolding equilibria were found to be independent of the fused partner. We conclude that the stabilizing effect SgsE_C exerts on EGFP is due to a reduction of degrees of freedom for folding of EGFP in the fused state.

• BMB Reports
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• 제37권6호
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• pp.741-748
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• 2004

The hepatitis C virus is associated with the development of liver cirrhosis and hepatocellular carcinomas. Among the 10 polyproteins produced by the virus, no function has been clearly assigned to the non-structural 5A (NS5A) protein. This study was designed to identify the hepatocellular proteins that interact with NS5A of the HCV. Yeast two-hybrid experiments were performed with a human liver cDNA prey-library, using five different NS5A derivatives as baits, the full-length NS5A (NS5A-F, amino acid (aa) 1~447) and its four different derivatives, denoted as NS5A-A (aa 1~150), -B (aa 1~300), -C (aa 300~447) and D (aa 150~447). NS5A-F, NS5A-B and NS5A-C gave two, two and 10 candidate clones, respectively, including an AHNAK-related protein, the secreted frizzled-related protein 4 (SFRP4), the N-myc downstream regulated gene 1 (NDRG1), the cellular retinoic acid binding protein 1 (CRABP-1), ferritin heavy chain (FTH1), translokin, tumor-associated calcium signal transducer 2 (TACSTD2), phosphatidylinositol 4-kinase (PI4K) and $centaurin{\delta}$ 2 ($CENT{\delta}2$). However, NS5A-A produced no candidates and NS5A-D was not suitable as bait due to transcriptional activity. Based on an in vitro binding assay, CRABP-1, PI4K, $CENT{\delta}2$ and two unknown fusion proteins with maltose binding protein (MBP), were confirmed to interact with the glutathione S-transferase (GST)/NS5A fusion protein. Furthermore, the interactions of CRABP-1, PI4K and $CENT{\delta}2$ were not related to the PXXP motif (class II), as judged by a domain analysis. While their biological relevance is under investigation, the results contribute to a better understanding of the possible role of NS5A in hepatocellular signaling pathways.

• KSBB Journal
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• 제23권3호
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• pp.205-212
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• 2008

본 연구의 목적은 위치특이적 펩타이드 및 단백질을 사용하여 재조합 대장균에서 활성형 인간 상피세포성장인자(hEGF)를 고효율로 발현할 수 있는 방법을 찾아내는 데 있다. 재조합 대장균내 cytoplasm 및 periplasm 영역에서 hEGF의 발현을 위해 각각 세개의 응합 펩타이드 및 단백질을 선정하여 상호 비교하였다. 재조합 대장균에서 hEGF의 발현유도시 대부분 불용성 단백질로 생산되는 현상을 극복하기 위해 cytoplasm영역에서는 ATS, thioredoxin, 리파제를 융합파트너로 사용하였으며 periplasm 영역에서는 foldase인 DsbA와 DsbC, 용융성 고발현 단백질인 maltose binding protein을 선택하여 사용하였다. Periplasm영역에서 발현유도를 시키는 융합단백질의 경우 cytoplasm영역에서의 발현양도 용융성 형태로 고발현 되는 것을 알 수 있었으며 전체적으로 약 2배가량의 용융성 형태로 발현되었다. hECF의 발현율을 가장 높일 수 있는 융합단백 질은 maltose binding protein이었으나 발현된 융합단백질의 24%가 불용성 단백질로 형성되어 활성형 형태로 얻는 데 한계가 있었으며, 활성형 형태로 hEGF의 발현을 위해서는 DsbA를 응합단백질로 사용한 경우에 18.1 mg/L로 가장 높은 발현농도를 보였다. Cytoplasm 영역에서 발현유도를 한 경우에는 ATS와 thioredoxin을 응합파트너로 hEGF를 발현한 경우 용융성 형태로 높은 발현율을 보였다. 특히 ATS와 같은 펩타이드를 N-말단에 융합시킨 경우 불용성을 방지하는 효과를 보여 이황화결합의 불완전성이나 소수성으로 인해 불용성 단백질로 발현되는 기존의 단백질을 활성형 형태로 발현하는데 될 수 있음을 확인할 수 있었다.

• Applied Biological Chemistry
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• 제38권2호
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• pp.111-117
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• 1995

CRP (cAMP receptor protein)은 cAMP와 결합하여 cAMP-CRP 복합체를 형성하여 전사조절의 조절인자로서 작용한다. crp 유전자에 변이를 도입하여 cAMP의 비존재 상태에서 cAMP-CRP와 비슷한 기능을 가진 crp 유전자가 도입된 대장균 MK2001 (crp, cya::km)을 숙주로 사용하여 cAMP 혹은 cGMP의 비존재하에서도 mal 유전자의 발현을 촉진시키는 유전자 sfs (sugar fermentation stimulation) 수 종을 클로닝 하였다. 본 실험에서는 이미 밝혀진 nlp (Ner like protein) 유전자와 같이, sfs의 새로운 유전자를 탐색하여, 그 중 sfs4의 2126 bp 전 염기배열을 결정하고, 잠정적인 sfs4의 promoter 영역에는 CRP 단백질과의 결합 DNA 공통 염기배열(5' AAT TGTGA ACACCA TCACC CGT 3')이 존재함을 확인했다. lacZ 융합 유전자를 작성하여 TP2010R1 MK2001의 균주에서 cAMP를 첨가할 경우 각각 2.3배, 1.8배의 ${\beta}-galactosidase$ 활성이 증가하는 것으로 보아 sfs4는 cAMP-CRP에 의해 발현 조절을 받는 것으로 나타났다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권3호
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• pp.155-162
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• 2002

The cyclic amidohydrolase family enzymes, which include allantoinase, dihydroorotase, dihydropyrimidinase and (phenyl)hydantoinase, are metal-dependent hydrolases and play a crucial role in the metabolism of purine and pyrimidine in vivo. Each enzyme has been independently characterized, and thus well documented, but studies on the higher structural traits shared by members of this enzyme family are rare due to the lack of comparative study. Here, we report upon the expression in E. coli cells of maltose-binding protein (MBP)- and glutathione S-transferase (GST)-fused cyclic amidohydrolase family enzymes, facilitating also for both simple purification and high-level expression. Interestingly, the native quaternary structure of each enzyme was maintained even when fused with MBP and GST. We also found that in fusion proteins the favorable biochemical properties of family enzymes such as, their optimal pHs, specific activities and kinetic properties were conserved compared to the native enzymes. In addition, MBP-fused enzymes showed remarkable folding ability in-vitro. Our findings, therefore, suggest that a previously unrecognized trait of this family, namely the ability to functional fusion with some other protein but yet to retain innate properties, is conserved. We described here the structural and evolutionary implications of the properties in this family enzyme.

• 한국동물위생학회지
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• 제34권3호
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• pp.201-208
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• 2011

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a pathogen of a variety of infections in horse. We studied biochemical and molecular characteristics of S. zooepidemicus isolated from the genital tract of Thoroughbred mares in Korea. Seventy-nine isolates were identified as S. zooepidemicus by biochemical and PCR method from 374 horses. The biochemical characteristics of S. zooepidemicus isolates were positive reaction of lactose and sorbitol. However, S. zooepidemicus isoltes were negative reaction of inulin, mannitol, raffinose, trehalose, aesculin hydrolysis, growth in 6.5% NaCl and variable reaction of maltose. Epidemiological investigations of S. zooepidemicus isolates were performed by fragment analysis of SzP (S. zooepidemicus protective protein) gene, CNE (collagen binding protein) gene and ISR (16s rRNA intergenic spacer region) gene using ABI Prism $3,130{\times}1$ Genetic Analyzer System. All isolates were shown single amplification size of 906 bp in CNE gene, but SzP and ISR gene were shown variable patterns of fragment size. The characteristics of S. zooepidemicus investigated in this study will be very useful for the prevention of infection and the studies of epidemiologic characteristics of S. zooepidemicus, causing the severe economic losses due to reproductive failures.

• 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.

• Journal of Microbiology and Biotechnology
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• 제26권3호
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• pp.603-609
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• 2016

Brucellosis affects a wide range of host species, including humans and many livestock animals. Chronic infections of the disease make antibiotic treatment costly, and the current vaccine used in livestock has not been approved for human use. This study investigated the possible use of the Brucella abortus outer membrane protein A (OmpA) as a candidate subunit vaccine in an infected mouse model. The ompA gene was cloned and overexpressed, and the recombinant OmpA (rOmpA) protein fused to maltose binding protein (MBP) was purified in Escherichia coli. Immunogenicity was verified through western blotting, and mice were immunized and challenged to evaluate its protective effect. Mice treated with rOmpA exhibited induced humoral and host cell-mediated responses, with a significant increase in immunoglobulin G (IgG1 and IgG2a) and cytokine levels, especially TNF-α and IL-12, compared with the control groups treated with either MBP or PBS. In conclusion, rOmpA should be highly considered as a future subunit vaccine for brucellosis, and further studies regarding rOmpA and its protective ability are suggested.