• Journal of Microbiology and Biotechnology
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• 제11권1호
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• pp.29-34
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• 2001

The mutant enzymes of N-carbamyl-D-amino aicd amidohydrolase (N-carbamylase) from Agrobacterium radiobacter NRRL B11291, showing a negligible activity, were selected from the library generated by random mutagenesis. From the sequence analysis, these mutants were found to contain the amino acids substitutions at Cys172, Glu47, and Glu146. Previously, Cys172 was reported to be necessary for the enzyme catalysis. The chemical modification of the N-carbamylase by carboxyl group specific chemical reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC), resulted in a loss of activity. The replacement of glutamic acids with glutamines by site-directed mutagenesis led to aggregation of the enzymes. Mutant enzymes fused with maltose binding protein (MBP) were expressed in soluble form, but were inactive. These results indicate that two glutamic acid residues play an important role in structure and function of the N-carbamylase. Multiple sequence alignment of the related enzymes revealed that Glu47 and Glu146 are rigidly conserved, which suggests that tese residues are crucial for the structure and function of the functionally related C-N hydrolases.

• Journal of Microbiology and Biotechnology
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• 제27권12호
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• pp.2156-2164
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• 2017

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as an antitumor agent owing to its ability to induce apoptosis of cancer cells without imparting toxicity toward most normal cells. TRAIL is produced in poor yield because of its insoluble expression in the cytoplasm of E. coli. In this study, we achieved soluble expression of TRAIL by fusing maltose-binding protein (MBP), b'a' domain of protein disulfide isomerase (PDIb'a'), or protein disulfide isomerase at the N-terminus of TRAIL. The TRAIL was purified using subsequent immobilized metal affinity chromatography and amylose-binding chromatography, with the tag removal using tobacco etch virus protease. Approximately 4.5 mg of pure TRAIL was produced from 125 ml flask culture with a purification yield of 71.6%. The endotoxin level of the final product was $0.4EU/{\mu}g$, as measured by the Limulus amebocyte lysate endotoxin assay. The purified TRAIL was validated and shown to cause apoptosis of HeLa cells with an $EC_{50}$ and Hill coefficient of $0.6{{\pm}}0.03nM$ and $2.41{\pm}0.15$, respectively. The high level of apoptosis in HeLa cells following administration of purified TRAIL indicates the significance and novelty of this method for producing high-grade and high-yield TRAIL.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권2호
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• pp.144-149
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• 2001

For the production and purification of a single chain human insulin precursor, four types of fusion peptides $\beta$-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)(sub)6-tagged sequence (HTS) were investigated. Recombinant E. coli harboring hybrid genes was cultivated at 37$\^{C}$ for 1h, and gene induction occurred when 0.2mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except for E. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0mM IPTG, followed by a longer than 4h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique.

• IMMUNE NETWORK
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• 제4권1호
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• pp.16-22
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• 2004

Background: To develop a novel treatment strategy for hepatitis B virus infection, a major cause of liver chirosis and cancer, we aimed to make human monoclonal antibodies inhibiting RNase H activity of P protein playing in important role in HBV replication. In this regard, phage display technology was employed and demonstrated as an efficient cloning method for human monoclonal antibody. So this study analysed the usability of human monoclonal antibody as protein based gene therapy. Methods: RNase H of HBV was expressed as fusion protein with maltose binding protein and purified with amylose resin column. Single chain Fv (scFv) phage antibody library was constructed by PCR cloning using total RNAs of PBMC from 50 healthy volunteers. Binders to RNase H were selected with BIAcore 2000 from the constructed library, and purified as soluble antibody fragment. The affinity and sequences of selected antibody fragments were analyzed with BIAcore and ABI automatic sequencer, respectively. And finally RNase H activity inhibiting assay was carried out. Results: Recombinant RNase H expressed in E. coli exhibited an proper enzyme activity. Naive library of $4.46{\times}10^9cfu$ was screened by BIAcore 2000. Two clones, RN41 and RN56, showed affinity of $4.5{\times}10^{-7}M$ and $1.9{\times}10^{-7}M$, respectively. But RNase H inhibiting activity of RN41 was higher than that of RN56. Conclusion: We cloned human monoclonal antibodies inhibiting RNase H activity of P protein of HBV. These antibodies can be expected to be a good candidate for protein-based antiviral therapy by preventing a replication of HBV if they can be expressed intracellularly in HBV-infected hepatocytes.

• Journal of Microbiology and Biotechnology
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• 제24권6호
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• pp.854-861
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• 2014

The diagnosis of Brucella abortus is mainly based on serological methods using antibody against LPS, which has diagnostic problems. Therefore, to solve this problem, we evaluated two proteins of B. abortus, Cu/Zn superoxide dismutase (SodC) and outer membrane proteins 2b porin (Omp2b). The genes were cloned and expressed in a pMAL system, and the recombinant proteins, rOmp2b and rSodC, were purified as fusion forms with maltose-binding protein. The identity of the proteins was confirmed by SDS-PAGE and Western blot analysis with sera of mice infected with B. abortus. Production of cytokines and nitric oxide (NO) was investigated in RAW 264.7 cells and mouse splenocytes after stimulation with the proteins. Moreover, cellular and humoral immune responses were investigated in BALB/c mice after immunization with the proteins. TNF-${\alpha}$, IL-6, and NO were significantly inducible in RAW 264.7 cells. Splenocytes of naive mice produced IFN-${\gamma}$ and IL-4 significantly by stimulation. Moreover, number of IgG, IFN-${\gamma}$, and IL-4 producing cells were increased in immunized mice with the two proteins. Production of IgG and IgM with rOmp2b was higher than those with rSodC in immunized mice. These results suggest that the two recombinant proteins of B. abortus may be potential LPS-free proteins for diagnosis.

• 생명과학회지
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• 제28권6호
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• pp.656-662
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• 2018

향상된 인디고이드 생산능력을 갖는 코리네박테리움 유래의 변이 플라빈-함유 모노옥시게나제(cFMO)를 개발하기 위하여, cFMO 효소의 상동성모델을 이용하여 말토오스-결합단백질(MBP)과 융합된 4가지 변이체(F170Y, A210G, A210S, T326S)를 제작하고 그 생화학적 특징을 밝혔다. 정제된 MBP-T326S는 최적 활성을 위하여 야생형보다 고농도의 FAD ($100{\mu}M$)를 요구하며, $100{\mu}M$의 FAD 첨가조건에서 $k_{cat}/K_m$이 3.8배 증가되었다. 인돌 옥시게나제 활성은 야생형의 63-77%를 나타냈다. MBP-T326S는 기질이 존재하지 않을 경우 쓸모없는 NADPH 산화효소 활성이 매우 낮은 수준을 보여주었다(21-24%). T326S이외의 변이 단백질들은 야생형에 비하여 $K_m$은 비슷하며 $k_{cat}/K_m$은 증가하였다. MBP-F170Y와 -A210S 변이단백질은 인돌 옥시게나제 활성이 각각 3.1배, 2.9배 증가하였다. 2.5 g/l의 트립토판을 함유한 LB배지에서 인디고이드 생산을 시험했을 때, 야생형 cFMO를 함유한 대장균은 684 mg/l의 인디고와 104 mg/l의 인디루빈을 생산한 반면, T326S를 함유한 세포는 1,040 mg/l의 인디고와 112 mg/l의 인디루빈을 생산하였다. 이전의 결과인 Methylophaga 유래의 FMO를 발현하는 대장균에서 가장 높은 수준인 920 mg/l의 인디고를 생산한 것과 비교하면, 본 연구결과는 인디고 생산이 13% 높은 수준이였다. 상동성 모델링에 기반한 cFMO의 단백질공학은 인디고이드 생산균을 개발하는데 보다 더 논리적인 전략을 제시하였다.

• 한국식품과학회지
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• 제46권1호
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• pp.73-78
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• 2014

본 연구는 돼지감자를 이용한 가공식품 개발 시 돼지감자에 대한 기초자료를 제공하고자 돼지감자의 영양성분 및 이화학적 특성을 분석하였다. 돼지감자의 수분함량은 $5.06{\pm}0.08%$, 조회분 $5.04{\pm}0.03%$, 조단백질은 $8.30{\pm}0.26%$, 조지방 $0.70{\pm}0.16%$, 탄수화물은 80.90%이었다. 돼지감자의 총당 함량은 $50.48{\pm}1.11$ mg/g 이었다. 색도를 측정한 결과 L값은 $94.16{\pm}0.03$, a값은 $0.32{\pm}0.01$, b값은 $0.30{\pm}0.01$로 측정되었다. 돼지감자의 수분결합능은 $4.04{\pm}0.16$ g/g, 수분활성도는 $0.245{\pm}0.005$였다. 돼지감자의 총 아미노산은 $1.337{\times}10^4$ mg/kg였고, 그 중 필수아미노산의 함량은 2,737 mg/kg였다. 아미노산 중 histidine이 7,305 mg/kg로 가장 많았고 leucine이 184.16 mg/kg로 가장 적은 양을 함유하고 있었다. 유리당은 fructose를 0.8%, maltose는 3.2% 함유하고 있었으며 지방산은 linoleic acid가 0.21%로 가장 높은 수치를 나타냈다. 무기질 함량은 K함량이 2,489 mg%로 가장 많이 함유되어 있었으며, 돼지감자의 비타민 C 함량은 $3.43{\pm}0.07$ mg%으로 측정되었다. 총 페놀 함량과 총 플라보노이드의 함량은 $3.06{\pm}0.07$ mg GAE/g, $1.89{\pm}0.03$ mg QE/g으로 나타났다.