• Plant Biotechnology Reports
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• 제3권2호
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• pp.157-165
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• 2009

Human erythropoietin (EPO) is a leading product in the biopharmaceutical market, but functional EPO has only been produced in mammalian cells, which limits its application and drives up the production costs. Using plants to produce human proteins may be an alternative way to reduce the cost. However, a recent report demonstrated that overexpression of the human EPO gene (EPO) in tobacco or Arabidopsis rendered males sterile and retarded vegetative growth, which raises concern whether EPO might interfere with hormone levels in transgenic plants. In the present study, we demonstrated that overexpressing EPO with additional 5'-His tag and 3' ER-retention peptides in tobacco did not cause any developmental defect compared to GUS plants. With our method, all 20 transgenic plants grew on selective medium and, further confirmed by PCR, were fertile. Most of them grew similarly compared to GUS plants. Only one transgenic plant (EPO2) was shorter in plant height but had twice the life span compared to other transgenic plants. When 11 randomly selected EPO plants, along with the abnormal plant EPO2, were subjected to RT-PCR analysis, all of them had detectable EPO transcripts. However, their protein levels varied considerably; seven of them had detectable EPO proteins analyzed by western blot. Our results indicate that overexpressing human EPO protein in plants does not have detrimental effects on growth and development. Our transformation systems allow us to further explore the possibility of glycoengineering tobacco plants for producing functional EPO and its derivatives.

• Journal of Microbiology and Biotechnology
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• 제15권3호
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• pp.652-655
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• 2005

This paper describes the development of an enzymatic assay system for the identification of inhibitors of isocitrate lyase (ICL), one of the key enzymes of the glyoxylate cycle that is considered as a new target for antifungal drugs. A 1.6 kb DNA fragment encoding the isocitrate lyase from Candida albicans ATCC10231 was amplified by PCR, cloned into a vector providing His-Patch-thioredoxin-tag at the N-terminus, expressed in Escherichia coli, and purified by metal chelate affinity chromatography. The molecular mass of the purified ICL was approximately 62 kDa, as determined by SDS-PAGE, and the enzyme activity was directly proportional to incubation time and enzyme concentration. The effects of itaconate-related compounds on ICL activity were also investigated. Among them, itaconic acid, 3-nitropropionate, and oxalate had strong inhibitory activities with $IC_{50}$ values of 5.8, 5.4 and $8.6\;{mu}g/ml$, respectively. These inhibitors also exhibited antifungal activity on YPD agar media containing acetate as a sole carbon source, albeit at high concentration. The results indicate that the C. albicans ICL may be a regulatory enzyme playing a crucial role in fungal growth and is a prime target for antifungal agents.

• Journal of Microbiology and Biotechnology
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• 제20권12호
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• pp.1689-1695
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• 2010

Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Here, we report the identification, cloning, and characterization of panK-sp from Streptomyces peucetius ATCC 27952. The gene encoded a protein of 332 amino acids with a calculated molecular mass of 36.8 kDa and high homology with PanK from S. avermitilis and S. coelicolor A3(2). To elucidate the putative function of PanK-sp, it was cloned into pET32a(+) to construct pPKSP32, and the PanK-sp was then expressed in E. coli BL21(DE3) as a His-tag fusion protein and purified by immobilized metal affinity chromatography. The enzyme assay of PanK-sp was carried out as a coupling assay. The gradual decrease in NADH concentration with time clearly indicated the phosphorylating activity of PanK-sp. Furthermore, the ca. 1.4-fold increase of DXR and the ca. 1.5-fold increase of actinorhodin by in vivo overexpression of panK-sp, constructed in pIBR25 under the control of a strong $ermE^*$ promoter, established its positive role in secondary metabolite production from S. peucetius and S. coelicolor, respectively.

• 한국통신학회논문지
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• 제38C권1호
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• pp.12-18
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• 2013

2012년 태그의 고유 식별 정보를 안전하게 숨기고, 매 세션 다른 값을 생성하기 위해서 해시함수와 AES 알고리즘을 모두 사용하는 DAP3-RS(Design of Authentication Protocol for Privacy Protection in RFID Systems)을 제안하였다. DAP3-RS 논문에서 Hash-Lock 프로토콜의 metaID가 고정되는 문제점을 AES(Advanced Encryption Standard) 알고리즘으로 해결하였고, 리더, 태그의 난수로 인증 과정을 거치기 때문에 스푸핑 공격, 재전송 공격, 트래픽 분석 등 다양한 공격에 안전하다고 주장하였다. 그러나 그의 주장과는 달리 고정된 해시 값으로 트래픽 분석이 가능하며, 리더와 태그사이의 동일한 데이터 값으로 인해 공격자임에도 불구하고 인증과정을 통과할 수 있다. 본 논문에서는 DAP3-RS가 공격자의 공격에 취약함을 증명한다. 그리고 AES 알고리즘 기반의 인증 프로토콜을 제안하고, 제안 프로토콜이 DAP3-RS에 비해 안전하고 효율적임을 증명한다.

• Journal of Microbiology and Biotechnology
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• 제22권12호
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• pp.1705-1713
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• 2012

A gene encoding ${\beta}$-glucosidase was cloned from Weissella cibaria 37, an isolate from human feces. Sequence analysis showed that the gene could encode a protein of 415 amino acids in length, and the translated amino acid sequence showed homology (34-31%) with glycosyl hydrolase family 1 ${\beta}$-glucosidases. The gene was overexpressed in E. coli BL21(DE3) using pET26b(+) and a 50 kDa protein was overproduced, which matched well with the calculated size of the enzyme, 49,950.87 Da. Recombinant ${\beta}$-glucosidase was purified by using a his-tag affinity column. The purified ${\beta}$-glucosidase had an optimum pH and a temperature of 5.5 and $45^{\circ}C$, respectively. Among the metal ions (5mM concentration), $Ca^{2+}$ slightly increased the activity (108.2%) whereas $Cu^{2+}$ (46.1%) and $Zn^{2+}$ (56.7%) reduced the activity. Among the enzyme inhibitors (1 mM concentration), SDS was the strongest inhibitor (16.9%), followed by pepstatin A (45.2%). The $K_m$ and $V_{max}$ values of purified enzyme were 4.04 mM and 0.92 ${\mu}mol/min$, respectively, when assayed using pNPG (p-nitrophenyl-${\beta}$-D-glucopyranoside) as the substrate. The enzyme liberated reducing sugars from carboxymethyl cellulose (CMC).

• The Plant Pathology Journal
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• 제27권1호
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• pp.37-43
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• 2011

Rice stripe virus (RSV) is one of serious epidemic pathogens for rice species grown in many Asian countries. Therefore, it is necessary to produce a diagnostic detection kit applicable in fields for RSV detection. In this study, RSV proteins that were derived from recombinant proteins and synthetic polypeptides as antigens were generated and were raised in rabbits for antiserum production. Among seven proteins in RSV, genes that code for NCP and NS3 proteins were cloned and subcloned into vector carrying His-tag protein and were expressed in E. coli. Of two recombinant proteins, only anti-NCP displayed stable hybridization signals in western blot analysis. Alternately, synthetic RSV polypeptides for CP, NCP, NS3 and NSvc4 we also generated and only antibodies against CP and NCP were very effective to detect RSV in both RSV infected rice and weed plants. However, antibodies against NS3 and NSvc4 showed weak specific bands as well as strong non-specific background due to the difference of viral proteins produced in the infected leaves. In summary, the antibodies generated against RSV proteins produced in this study will be useful for various assays such as for RSV diagnostic detection, immunoprecipitation, protein purification, and western blot analysis.

• 한국자기공명학회논문지
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• 제17권2호
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• pp.67-75
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• 2013

ATP synthase produces ATP, a major energy source for metabolic processes in organisms, from ADP and inorganic phosphate in cellular membranes. ATP synthase is known as a rotary motor, in which the c-subunit ring functions as a rotor. In this work, we have tried to develop a more general preparation procedure of thermophilic $F_oc$-ring ($TF_oc$-ring) for NMR measurements. The expression of $TF_oF_1$ is easily affected by various experimental conditions such as temperature, shape and size of a flask, a volume of medium, and shaking rate of an incubator. Accordingly, we have tried to optimize the expression conditions of $TF_oF_1$. $TF_oc$-rings were purified from $TF_oF_1$ according to a reported method. We modified purification procedures to improve purity and yield of $TF_oc$. On top of them, we found a new combination of detergents for the purification at anion-exchange column chromatography. To examine the effect of lipid environments on the structure, the $TF_oc$-rings were reconstituted into two kinds of lipid bilayers, namely, saturated and unsaturated lipid ones. Then, we have compared characteristics of the $TF_oc$-ring structures in these membranes with solid-state NMR.

• Journal of Microbiology and Biotechnology
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• 제29권9호
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• pp.1383-1390
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• 2019

In this study, we expressed cotA laccase from Bacillus subtilis on the surface of B. subtilis spores for efficient decolorization of synthetic dyes. The cotE, cotG, and cotY genes were used as anchoring motifs for efficient spore surface display of cotA laccase. Moreover, a $His_6$ tag was inserted at the C-terminal end of cotA for the immunological detection of the expressed fusion protein. Appropriate expression of the CotE-CotA (74 kDa), CotG-CotA (76 kDa), and CotY-CotA (73 kDa) fusion proteins was confirmed by western blot. We verified the surface expression of each fusion protein on B. subtilis spore by flow cytometry. The decoloration rates of Acid Green 25 (anthraquinone dye) for the recombinant DB104 (pSDJH-EA), DB104 (pSDJH-GA), DB104 (pSDJH-YA), and the control DB104 spores were 48.75%, 16.12%, 21.10%, and 9.96%, respectively. DB104 (pSDJH-EA) showed the highest decolorization of Acid Green 25 and was subsequently tested on other synthetic dyes with different structures. The decolorization rates of the DB104 (pSDJH-EA) spore for Acid Red 18 (azo dye) and indigo carmine (indigo dye) were 18.58% and 43.20%, respectively. The optimum temperature for the decolorization of Acid Green 25 by the DB104 (pSDJH-EA) spore was found to be $50^{\circ}C$. Upon treatment with known laccase inhibitors, including EDTA, SDS, and $NaN_3$, the decolorization rate of Acid Green 25 by the DB104 (pSDJH-EA) spore decreased by 23%, 80%, and 36%, respectively.

• Journal of Microbiology and Biotechnology
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• 제32권8호
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• pp.1041-1046
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• 2022

Nucleoside deoxyribosyltransferase (NDT) is an enzyme that replaces the purine or pyrimidine base of 2'-deoxyribonucleoside. This enzyme is generally used in the nucleotide salvage pathway in vivo and synthesizes many nucleoside analogs in vitro for various biotechnological purposes. Since NDT is known to exhibit relatively low reactivity toward nucleoside analogs such as 2'-fluoro-2'-deoxynucleoside, it is necessary to develop an enhanced NDT mutant enzyme suitable for nucleoside analogs. In this study, molecular evolution strategy via error-prone PCR was performed with ndt gene derived from Lactobacillus leichmannii as a template to obtain an engineered NDT with higher substrate specificity to 2FDU (2'-fluoro-2'-deoxyuridine). A mutant library of 214 ndt genes with different sequences was obtained and performed for the conversion of 2FDU to 2FDA (2'-fluoro-2'-deoxyadenosine). The E. coli containing a mutant NDT, named NDT^L59Q, showed 1.7-fold (at 40℃) and 4.4-fold (at 50℃) higher 2FDU-to-2FDA conversions compared to the NDT^WT, respectively. Subsequently, both NDT^WT and NDT^L59Q enzymes were over-expressed and purified using a His-tag system in E. coli. Characterization and enzyme kinetics revealed that the NDT^L59Q mutant enzyme containing a single point mutation of leucine to glutamine at the 59^th position exhibited superior thermal stability with enhanced substrate specificity to 2FDU.

• Journal of Applied Biological Chemistry
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• 제65권4호
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• pp.383-386
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• 2022

Thermotoga maritima cellulose B 유전자의 The open reading frame (ORF)은 825 bp이고, cellulase B는 분자량이 약 31,732 kDa인 274개의 아미노산으로 구성되어 있다 이 중 N-말단의 17개 아미노산은 signal peptide로 추정된다. Signal peptide를 제외한 ORF를 pRSET 대장균 발현벡터에 삽입하여 pRBTmCelB를 제조하였다. 6-His tag을 포함하는 TmCelB 융합단백질의 cellulose 분해활성과 생화학적 특성을 분석하기 위해 pRBTmCelB를 대장균 BL21에서 과다발현하였고 순수분리하였다. TmCelB 융합단백질은 cellulose 분해활성을 나타내었고 이 있는 것으로 분석되었다. TmCelB 융합단백질은 약 95 ℃ 부근에서 가장 높은 효소 활성을 나타내었고, 100 ℃에서 최대 활성을 80% 이상 유지하는 것을 확인하였다. 또한 TmCelB 융합단백질은 약 pH 4.5 부근에서 최대 활성을 나타내었고, pH 4.0-6.0 범위에서 최대 활성을 60% 이상 유지하였다. TmCelB 융합단백질은 100 ℃에서 180분 후에도 효소활성을 80% 이상 유지하였다.