• Journal of Microbiology and Biotechnology
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• 제23권5호
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• pp.689-698
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• 2013

The production and characterization of an active recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21(DE3) has been previously reported. In this study, the effect of the signal peptide (SP), inducer concentration, process scale, and operational mode (batch and semi-continuous) on GALNS production were evaluated. When native SP was presented, higher enzyme activity levels were observed in both soluble and inclusion bodies fractions, and its removal had a significant impact on enzyme activation. At shake scale, the optimal IPTG concentrations were 0.5 and 1.5 mM for the strains with and without SP, respectively, whereas at bench scale, the highest enzyme activities were observed with 1.5 mM IPTG for both strains. Noteworthy, enzyme activity in the culture media was only detected when SP was presented and the culture was carried out under semi-continuous mode. We showed for the first time that the mechanism that in prokaryotes recognizes the SP to mediate sulfatase activation can also recognize a eukaryotic SP, favoring the activation of the enzyme, and could also favor the secretion of the recombinant protein. These results offer significant information for scaling-up the production of human sulfatases in E. coli.

• Journal of Microbiology and Biotechnology
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• 제16권6호
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• pp.901-910
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• 2006

Poly(3-hydroxybutyrate) [P(3HB)] is a microbial polyester intracellularly accumulated as distinct granules in numerous microorganisms as an energy and carbon storage material. Recombinant Escherichia coli harboring the heterologous P(3HB) biosynthesis genes accumulates large amounts of P(3HB) granules, yet the granule-associated proteins have not been identified. Therefore, this study reports on an analysis of the P(3HB) granule-associated proteome in recombinant E. coli. Fiye proteins out of 7 spots identified were found to be involved in functions of translation, heat-stress responses, and P(3HB) biosynthesis. Two of the major granule-associated proteins, IbpA/B, which are already known to bind to recombinant proteins forming inclusion bodies in E. coli, were further analyzed. Immunoblotting and immunoelectron microscopic studies with IbpA/B antibodies clearly demonstrated the binding and localization of IbpA/B to P(3HB) granules. IbpA/B seemed to play an important role in recombinant E. coli producing P(3HB) by stabilizing the interface between the hydrophobic P(3HB) granules and the hydrophilic cytoplasm. Thus, IbpA/B were found to act like phasins in recombinant E. coli, as they are the major proteins bound to the P(3HB) granules, affect the morphology of the granules, and reduce the amount of cytosolic proteins bound to the P(3HB) granules.

• 대한바이러스학회지
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• 제28권1호
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• pp.31-38
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• 1998

In Korea, all domestic made test systems for detecting antibodies in HIV-1 contain the antigens from human immunodeficiency type 1 (HIV-1) subtype B. However, because HIV-1 subtype O is significantly different in amino acid sequences from all other subtypes of HIV-1, there has been a need for developing a test for detecting antibodies in subtype O. For this purpose, the entire nucleotide sequence corresponding to the extracellular domain of the transmembrane glycoprotein of HIV-1 subtype O was synthesized with consideration of Escherichia coli condon usage. Various regions of the extracellular domain were cloned into E. coli expression vectors and tested for levels of protein production. The nucleotide sequence, named ECTM, that can encode a 129 amino acid-long peptide, was found to be expressed at a high level in E. coli. The protein of approximately 17 kDa specifically reacted with sera from individuals infected with HIV-1 subtype O. The ECTM protein was purified to near homogeneity by the CM-T gel chromatography, using concentrated, denatured inclusion bodies. In Western blot analysis, the purified viral antigen reacted with sera from individuals infected with subtype O more efficiently than subtype B. The enzyme linked immunoabsorbent assay (ELISA) system was developed using the subtype O viral protein and compared with the commercially available kit lacking the antigens from subtype O. The ELISA kit containing the subtype O antigen ECTM alone efficiently reacted with sera from individuals infected with subtype O. The subtype O antigen-containing kit produced a positive absorbence even when sera were diluted 512-fold, suggesting a high sensitivity. The commercially available kit also reacted with subtype O sera, but produced a negative result at a dilution of 8-fold. Our results suggest that the currently available kit may not be able to efficiently detect subtype O sera and that the viral protein developed in this study may be added to the current system to maximize the detection of sera from individuals infected with subtype O.

• 한국응용곤충학회지
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• 제20권1호
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• pp.6-14
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• 1981

모자익 Virus병에 감염된 마늘인편에 열처리, 화학치료제의 효과를 본 시험결과는 다음과 같다. 수중 또는 기중 열처리에 있어서 공히 $37^{\circ}\~57^{\circ}C$에서 35일간-1시간의 처리로서는 마늘 Virus의 불활성 화효과가 없었고, $62^{\circ}\~72^{\circ}C$에 $90\~5$분간의 처리로서 마늘엽에 나타난 병징이 감소되기는 하였으나 식물체의 생장력이 매우 약화되었으므로 열처리로서는 마늘 Virus를 완전히 불활성화 시킬 수 없었다. 마늘 인편을 $10\~50ppm$의 Malachite Green, 동농도의 2,4-Dichlorophenoxy Acetic Acid 또는 $20\~100ppm$의 Quinhydrone 수용액에 24시간 침윤처리한 후에 식재생장한 마늘엽에 모자익 병징은 감소되었으나 고농도에서 마늘의 생육이 억제되었다. $10\~50ppm$의 Naphthyl Activ A챵에 침윤한 마늘 인편을 식재 하였을 때에도 생장한 마늘엽에 모자익 병징이 감소되기는 하였으나 완전히 없어지지는 않았다. 수정한 Murashigh-Skoog 배지에 $0.5\~1.5ppm$의 Naphthyl Acetic Acid를 가용하여 마늘의 조직을 배양하였을 때에는 신생한 마늘식생체내에 Virus가 불활성화되어 엽에는 모자익 병징이 나타나지 않았으며 조직세포내에는 봉입체가 형성되지 않고, 정상적인 핵을 가진 건전한 식물체를 얻을 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제17권4호
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• pp.638-643
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• 2007

The full encoding sequence for human type II hexokinase (HXK II) was cloned into the E. coli expression vector pET 21b and expressed as a C-terminally hexahistidine-tagged protein in the BL2l (DE3) strain. The IPTG-induced HXK II approximately accounted for 17% of the total E. coli proteins, and 81% of HXK $II_{6{\times}His}$ existed in inclusion bodies. To improve the production of soluble recombinant HXK II protein, in the functionally active form, we used low temperature, and the osmotic stress expression method. When expressed at $18^{\circ}C$, about 83% of HXK $II_{6{\times}His}$ existed in the soluble fraction, which amounted to a 4.1-fold yield over that expressed at $37^{\circ}C$. The soluble form of HXK $II_{6{\times}His}$ was also highly produced in the presence of 1M sorbitol under the standard condition $(37^{\circ}C)$, which indicated that temperature downshift and low water potentials were required to improve the yield of active recombinant HXK II protein. The expressed protein was purified by metal chelate affinity chromatography performed in an IDA Excellose column charged with $Ni^{2+}$ ions, resulting in about 40mg recombinant HXK II protein obtained with purity over 89% from 51 of E. coli culture. The identity of HXK $II_{6{\times}His}$ was confirmed by Western blotting analysis. Taken together, using the stress-governed expression described in this study, human active HXK II can be purified in sufficient amounts for biochemical and biomedical studies.

• Journal of Microbiology and Biotechnology
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• 제28권10호
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• pp.1749-1759
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• 2018

Recombinant (rec) prion protein (PrP) is an extremely useful resource for studying protein misfolding and subsequent protein aggregation events. Here, we report mass production of high-purity rec-polypeptide encoding the C-terminal globular domain of PrP; (90-230) for human and (89-231) for murine PrP. These proteins were expressed as His-tagged fusion proteins in E. coli cultured by a high cell-density aerobic fermentation method. RecPrPs recovered from inclusion bodies were slowly refolded under reducing conditions. Purification was performed by a sequence of metal-affinity, cation-exchange, and reverse-phase chromatography. The current procedure yielded several dozens of milligrams of recPrP per liter with >95% purity. The purified recPrPs predominantly adopted an ${\alpha}$-helix-rich conformation and were functionally sufficient as substrates to measure the seeding activity of human and animal prions. Establishment of a procedure for high-level production of high-purity recPrP supports the advancement of in vitro investigations of PrP including diagnosis for prion diseases.

• 식물병연구
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• 제8권2호
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• pp.92-100
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• 2002

한국산 땅콩에 감염되어 있는 바이러스를 동정하기 위하여 땅콩 재배지 에서 모자이크, 괴저를 동반한 얼룩무늬, 황화, 줄무늬, 엽맥녹대, 위축 등의 바이러스 감염 증상을 나타내는 땅콩잎 및 땅콩을 채집하였다. 이들 시료로부터 기주범위, 면역전자현미경(ISEM), 감염 세포내의 바이러스의 존재양식, direct immune staining assay(DISA), RT-PCR 등에 의하여 Bean common mosaic virus(BCMV-PSt)와 Peanut mottle virus(PeMoV)를 분리하였다. 이들 시료를 DN법에 의거하여 전자현미경으로 관찰한 바 길이가 약 780 nm의 사상형 입자와 세포질 봉입체를 관찰하였다. 초박절편의 시료 관찰에서도 길이 약 700 nm의 사상 입자가 엽육세포 등의 세포질과 액포에 산재 혹은 병행 배열로 존재해 있는 영상 및 세포질 봉입체가 반드시 확인되었다. 또한, 이들 시료를 BCMV-PSt와 PeMoV의 항혈청으로 ISEM을 실시한 결과, 두 항체에 모두 decoration 되었음을 확인하였다. 두 바이러스가 종자전염이 되는 지를 확인하기 위하여 땅콩을 직접 BCMV-PSt와 PeMoV의 항혈청으로 DISA를 실시하였다. 그 결과, BCMV-PSt 및 PeMoV 항혈청에 발색이 되었으며, 이들을 발아시켜 유식물체를 획득하였다. 이들 유식물체에서도 바이러스 감염 증상인 얼룩무늬 증상이 관찰되었고, 이들을 BCMV-PSt와 PeMoV의 항혈청으로 ISEM방법으로 검경한 결과, 두 항체에 모두 decoration 되었다. 또한, 자연감염 식물체 및 DISA에 의해 바이러스 감염이 확인된 종자를 발아시킨 2년생 식물체로부터 생물검정 법 및 ISEM에 의해 두 종의 바이러스가 검출되었다. Rl-PCR을 실시한 결과, 약 1.2Kb크기의 BCMV-PSt coat protein유전자가 증폭 되었다. 이 연구결과, 한국산 땅콩에 BCMV-PSt가 가장 많이 감염되어 있음을 확인하였다.

• Applied Microscopy
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• 제12권2호
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• pp.55-68
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• 1982

The study on the nerve cells in the pars intercerebralis(IP) of 5-day-old cabbage butterfly Pieris rapae L. was performed to observe their ultrastructures and classify them on the basis. of the differences in size, shape and relative distribution cf cell organelles. The brain-subesophageal ganglion complex was fixed in 1% paraformaldehyde-1% gluaraldehyde mixture and embedded in araldite mixture. The transverse thin sections of IP were stained with uranyl acetate and lead citrate and examined by Hitachi 500 and ]EM 100B electron microscope. Five distinct types. of nerve cells are recognized and are arbitrarily designated as Type I, Type II Type III, Type IV and Type V. Type I neurone: These neurones are neurosecretory cells. Several neurosecretory cells are. recognized in the pars intercerebralis. They are roughly round or peach-shaped cells measuring $13{\sim}25{\mu}m$ in diameter. The rounded nucleus shows about $5{\sim}10{\mu}m$ in diameter. The chromatin is predominantly diffused with only occasional dense patches. The perikaryon contains numerous. mitochondria, free polyribosomes and neurosecretory granules. The neurosecretory granules are relatively uniform in electron density, and each one is about $100{\sim}400{\mu}m$ in diameter and surrounded by a single membrane. The granules are also observed mostly as in groups. In one group of neurones the cisternae of endoplasmic reticulum are distended or in other group of neurones are not distended. Golgi saccules are slightly dilated at their lateral extremities and contains. frequenty dense rounded materials. Type II neurone: Thes have the largest soma in the pars intercerebralis about $30{\sim}35{\mu}m$ in diameter. They also show roughly polygonal in shape. The nucleus is elongated or sickle-shaped. The chromatin is mainly in the euchromatin form. The perikarya in these cells are well populated with populated with free ribosomes and contain numerous mitochondria and Golgi bodies. The cisternae of granular endoplasmic reticulum are also well distributed. Type III neurone: They are oval or spindle-shaped and also medium-sized. neurones approximately $15{\sim}17{\mu}m$ in length. The nucleus is oval or slightly elongated in shape and $8{\sim}9{\mu}m$ in length. The chromatin occurs in diffused form. The cytoplasm contains many filamentous or oval mitochondria. The perikaryon has also numerous free polyribosomes and cisternae of granular endoplasmic reticulum. Type VI neurone: They are roughly polygonal in shape probably due to the close approximation of the adjacent cells. The soma is about $7{\sim}8{\mu}m$ in diameter. The nucleus is round or oval in shape and $5.0{\sim}5.8{\mu}m$ in diameter. The necleus also occupies a large proprion of the cell body. The perikaryon is well populated with free ribosomes and contains several mitochondria and cistenae of granular endoplasmic reticulum. Type V neurone: These neurones are similar to Type VI neurones in various respects such as cell size and cell inclusion, but they differ from Type IV neurones in shape. The soma is oval or slightly elongated. The cell body contains several filamentous and oval mitochondria.

• 한국임상수의학회지
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• 제30권3호
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• pp.210-213
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• 2013

고양이 헤르페스 바이러스-1 감염과 관련된 고양이의 괴사성 안면 피부염의 증례를 보고하고자 한다. 3살된, 미거세 수컷 도메스틱 숏헤어 고양이가 2년간 지속된 소양성 피부염을 주증으로 내원하였다. 환자는 생후 약 2개월령부터 지속적인 눈꼽과 결막염을 가지고 있었으며, 눈 주위부터 안면 전체로 확대된 피부염의 병력을 가지고 있었다. 내원 당시에는 가피로 덮여있는 미란과 궤양성의 피부병변이 안면과 회음부에서 주로 관찰되었다. 피부 병변부위의 조직학적 검사 결과, 경계면 피부염을 동반한 기저층의 수포변성과 각질세포의 단세포 괴사 소견이 관찰되었으며, 호산구 침윤을 동반한 표피와 진피의 괴사와 각질세포의 핵내 봉입체도 관찰되었다. 이에 가피 부위의 가검물을 이용하여 PCR검사를 수행하였고, 그 결과 고양이 헤르페스 바이러스-1의 유전자가 검출되었다. 위의 소견으로부터, 본 증례는 고양이 헤르페스 바이러스-1 감염과 관련된 궤양성 피부염으로 진단되었고, 아시클로버의 경구투여와 고양이 인터페론 오메가의 국소투여로 피부 및 점막 병변의 뚜렷한 개선을 볼 수 있었다.

• 생명과학회지
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• 제17권1호
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• pp.132-136
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• 2007

E. coli에서 Pseudoalteromonas elyakovii 유래의 alginate lyase유전자(aly)를 발현시킬 때, 대부분의 단백질이 불용성 내포체 형태로 발현됨을 확인하였다. Alginate lyase를 가용성 활성형으로 생산하기 위해 aly와 DnaK/DnaJ/GrpE 또는 aly와 GroEL/ES을 공발현하는 형질전환체를 얻었다. 공발현 결과, 단백질의 올바른 접힘을 도와주는 DnaK/DnaJ/GrpE chaperone이 가용성 및 활성형의 alginate lyase 생산에 매우 효과적임을 알 수 있었다. DnaK/DnaJ/GrpE chaperone의 발현에 유도제인 L-arabinose 최적 농도는 0.05 mg/ml이었으며, 이러한 공발현에 의해 약 34%의 alginate lyase가 가용성 분획에서 생산되었다. 또한 10%의 cetylpyridinium chloride를 첨가함으로써, 공발현 콜로니 주위에 투명환이 형성됨을 확인할 수 있었고, 이는 활성형 alginate lyase 효소에 의해 alginate가 분해되었음을 시사하였다.