• 생물정신의학
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• 제8권1호
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• pp.62-70
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• 2001

Alzheimer's disease(AD) is associated with a characteristic neuropathology. The major hallmarks of AD are senile plaques (SPs) and neurofibrillary tangles(NFTs). ${\beta}$-amyloid protein($A{\beta}$) is derived from the proteolysis of amyloid precursor protein(APP) and then converted to SPs. Mature SPs produce cytotoxicity through direct toxic effects and activation of microglia and complement. NFTs are composed of paired helical filaments(PHFs) including abnormally phosphorylated form of the microtubule-associated protein(MAP) tau and increased tau level in cerebrospinal fluid may be observed in most AD. The aggregation of $A{\beta}$ and tau formation are thought to be a final common pathway of AD. Acetylcholine, dopamine, serotonin, GABA and their receptors are associated with AD. Especially, decreased nicotinic acetylcholine receptors(nAChRs) in AD are reported. Genetic lesions associated with AD are mutations in the structural genes for the APP located on chromosome 21, presenilin(PSN)1 located on chromosome 14 and PSN2 located on chromosome 1. Also, trisomy 21, Apo-E gene located on chromosome 19, PMF locus, low density lipoprotein receptor-related protein and ${\alpha}$-macroglobulin increase risk of AD. In this article, we will review about the neurobiology of AD and some newly developed research areas.

• Applied Biological Chemistry
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• 제51권4호
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• pp.276-280
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• 2008

식품 allergen 저감화 수단으로 자외선 조사의 타당성을 검토하고 자외선조사가 식품 단백질의 분자적 성질에 미치는 영향을 조사하고자 ovalbumin 용액에 자외선을 조사한 후 단백질의 분자량 분포와 2차구조 및 3차구조의 변화를 조사하였다. SDS-PAGE와 Gel permeation chromatography 결과 ovalbumin은 자외선 조사에 의하여 단백질이 분해되고 조사시간이 증가할수록 펩티드가 중합하는 형태를 나타냈다. Circular dichroism 연구는 자외선 조사에 의하여 ${\alpha}$-helix 구조가 감소하고 조사시간이 증가할 경우 compact denatured ovalbumin의 구조를 나타내는 2차구조의 변화를 나타냈다. 자외선 조사된 ovalbumin의 형광스펙트럼은 조사시간이 증가할수록 단백질의 maximum emission intensity가 감소하는 3차구조의 변화를 나타냈다. 결과적으로 자외선 조사는 ovalbumin의 분자적 성질을 변화시키며 allergen의 항원성을 변화시키는 수단으로 이용가능성을 시사한다.

• Journal of Microbiology and Biotechnology
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• 제17권11호
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• pp.1751-1757
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• 2007

Elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition upon a change in temperature. This thermally triggered phase transition allows for a simple and rapid means of purifying a fusion protein. Recovery of ELPs-tagged fusion protein was easily achieved by aggregation, triggered either by raising temperature or by adding salt. In this study, levansucrase has been used as a model enzyme in the development of a simple one-step purification method using ELPs. The levansucrase gene cloned from Pseudomonas aurantiaca S-4380 was tagged with various sizes of ELPs to functionally express and optimize the purification of levansucrase. One of two ELPs, ELP[V-20] or ELP[V-40], was fused at the C-terminus of the levansucrase gene. A levansucrase-ELP fusion protein was expressed in Escherichia coli $DH5{\alpha}$ at $37^{\circ}C$ for 18 h. The molecular masses of levansucrase-ELP[V-20] and levansucrase-ELP[V-40] were determined as 56 kDa and 65 kDa, respectively. The phase transition of levansucrase-ELP[V-20] occurred at $20^{\circ}C$ in 50 mM Tris-Cl (pH 8) buffer with 3 M NaCl added, whereas the phase transition temperature ($T_t$) of levansucrase-ELP[V-40] was $17^{\circ}C$ with 2 M NaCl. Levansucrase was successfully purified using the phase transition characteristics of ELPs, with a recovery yield of higher than 80%, as verified by SDS-PAGE. The specific activity was measured spectrophotometrically to be 173 U/mg and 171 U/mg for levansucrase-ELP[V-20] and levansucrase-ELP[V-40], respectively, implying that the ELP-tagging system provides an efficient one-step separation method for protein purification.

• Journal of Microbiology and Biotechnology
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• 제19권1호
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• pp.72-77
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• 2009

Here, we demonstrate that the overexpression of the GroELS chaperone system, which assists the folding of intracellular proteins and prevents aggregation of its biological targets, can enhance the thermotolerance of Escherichia coli strains and facilitate the production of recombinant protein under thermal stress. The overexpression of GroELS led to an about 2-fold higher growth rate of E. coli XL-1 blue than control at $45^{\circ}C$ and induced the growth of the strain even at $50^{\circ}C$, although the growth was not sustained in the second-round culture. The effect of GroELS overexpression was also effective on other E. coli strains such as JM109, $DH5{\alpha}$, and BL21. Finally, we have shown that coexpression of GroELS allows us to produce recombinant protein even at $50^{\circ}C$, a temperature at which the protein production based on E. coli is not efficient. This study indicates that the employment of the GroELS overexpression system can expand the range of environmental conditions for E. coli.

• KSBB Journal
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• 제16권2호
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• pp.146-152
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• 2001

rhGH-GST 융합단백질을 사용하여 재조합 대장균 세포 파쇄액으로부터 직접적으로 내포체의 solid-phase 재접힘을 수행할 수 있는 새로운 공정을 개발하였다. 그것은 고체 업자를 제거하는 동시에 초기에 목적딴백질을 흡착 포집할 수 있 는 expanded bed adsorption 크로마토그래피의 장점을 이용한 것이다. 세포 파쇄액 내 용해훤 내포체로부터의 풀린 융합단백질은 expanded bed adsorption 원리에 의해 STREAMLINE DEAE resin에 흡착되고 세포 찌꺼기 등 고체 입자물들은 위 방향 흐름에 의해 효과적으로 제거된다. Urea를 접차적으로 제거함으로써 융합단백질은 고체 matrix 표면에서 재접힘 된 후 염 놓도 구배에 의해 용출된다. 이 새로운 EBA-mediat$\xi$d 재접힘 방법은 응집현상을 획기적으로 줄이고 공정수율윤 향상시킬 뿐 아나라 공정단계 수를 줄일 수 있다. 이 공정은 우리가 알고 있는 한 세계에서 최초로 개발된 공정이며, 현재 single-chain polypeptide, affinity-tagged protein 등과 갈은 다른 행태의 단백질에 EBA를 사용한 재접힘 공정올 적용시키가 위한 연구가 진행되고 있다.

• Journal of Microbiology and Biotechnology
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• 제34권3호
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• pp.525-537
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• 2024

Pilins are protein subunits of pili. The pilins of type IV pili (T4P) in pathogenic bacteria are well characterized, but anything is known about the T4P proteins in acidophilic chemolithoautotrophic microorganisms such as the genus Acidithiobacillus. The interest in T4P of A. thiooxidans is because of their possible role in cell recruitment and bacterial aggregation on the surface of minerals during biooxidation of sulfide minerals. In this study we present a successful ad hoc methodology for the heterologous expression and purification of extracellular proteins such as the minor pilin PilV of the T4P of A. thiooxidans, a pilin exposed to extreme conditions of acidity and high oxidation-reduction potentials, and that interact with metal sulfides in an environment rich in dissolved minerals. Once obtained, the model structure of A. thiooxidans PilV revealed the core basic architecture of T4P pilins. Because of the acidophilic condition, we carried out in silico characterization of the protonation status of acidic and basic residues of PilV in order to calculate the ionization state at specific pH values and evaluated their pH stability. Further biophysical characterization was done using UV-visible and fluorescence spectroscopy and the results showed that PilV remains soluble and stable even after exposure to significant changes of pH. PilV has a unique amino acid composition that exhibits acid stability, with significant biotechnology implications such as biooxidation of sulfide minerals. The biophysics profiles of PilV open new paradigms about resilient proteins and stimulate the study of other pilins from extremophiles.

• 한국식품영양과학회지
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• 제31권5호
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• pp.723-727
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• 2002

감마선 조사에 의 한 마늘의 주요 알러젠 단백질인 allivin의 구조적 변화를 관찰하였다. Allivin 용액 (3.0 mg/mL in 0.01 M PBS, UH 7.4)과 생 마늘에 2.5, 5.0, 7.5, 10.0 kGy의 흡수선량을 갖도록 감마선을 조사하였다. 조사 후 용액의 절반을 0.22$\mu\textrm{m}$ filter로 여과하여 준비하였다. 감마선 조사에 의한 allivin의 변화는 UV spectrum과 SDS-PAGE를 사용하여 관찰하였다. 가시적으로 관찰하였을 때, 조사선량이 증가할수록 allivin 용액의 혼탁도가 증가하였으며 ,660 nm 파장에서의 흡광도의 증가로 확인할 수 있었다. 조사 후 여과한 allivin 용액의 UV spectrum 측정에서 혼탁도의 변화가 크게 감소되었으며, 선량이 증가할수록 용해된 단백질의 농도가 소하였다. SDS-PAGE시험에서 감마선 조사된 allivin의 큰 변화는 관찰되지 않았으나, 분자간 응집이 관찰되었다 그러나, 조사 후 여과한 시료에서 는 allivin의 감소가 관찰되었으며, 분자간 응집으로 발생된 끌림도 감소하였다. 생마늘에 대한 전기영동 결과는 차이가 없었다. 이 결과들은 감마선 조사가 수용액 상태에서 allivin의 알러지성을 감소시킬 수 있음을 시사한다.

• BMB Reports
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• 제44권12호
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• pp.816-820
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• 2011

There is a broad range of different small heat shock proteins (sHSPs) that have diverse structural and functional characteristics. To better understand the functional role of mitochondrial sHSP, NtHSP24.6 was expressed in Escherichia coli with a hexahistidine tag and purified. The protein was analyzed by non-denaturing PAGE, chemical cross-linking and size exclusion chromatography and the $H_6NtHSP24.6$ protein was found to form a dimer in solution. The in vitro functional analysis of $H_6NtHSP24.6$ using firefly luciferase and citrate synthase demonstrated that this protein displays typical molecular chaperone activity. When cell lysates of E. coli were heated after the addition of $H_6NtHSP24.6$, a broad range of proteins from 10 to 160 kD in size remained in the soluble state. These results suggest that NtHSP24.6 forms a dimer and can function as a molecular chaperone to protect a diverse range of proteins from thermal aggregation.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2005년도 2005 Annual Meeting & International Symposium
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• pp.230-232
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• 2005

The formation of insoluble aggregation of the recombinant kringle fragment of human apolipoprotein(a), rhLK8, in endoplasmic reticulum was identified as the rate-limiting step in the rhLK8 secretion in Saccharomyces cerevisiae. To analyze the protein secretion pathway, some of yeast genes closely related to protein secretion was rationally selected and their oligomer DNA were arrayed on the chip. The expression profiling of these genes during the induction of rhLK8 in fermentor fed-batch cultures revealed that several foldases including pdi1 gene were up-regulated in the early induction phase, whereas protein transport-related genes were up-regulated in the late induction phase. The coexpression of pdi1 gene increased rhLK8-folding capacity. Hence, the secretion efficiency of rhLK8 in the strain overexpressing pdi1 gene increased by 2-fold comparing in its parental strain. The oligomer DNA chip arrayed with minimum number of the genes selected in this study could be generally applicable to the monitoring system for the heterologous protein secretion and expression in Saccharomyces cerevisiae. With the optimization of fed-batch culture conditions and the alteration of genetic background of host, we obtained extracellular rhLK8 at higher yields than with Pichia pastoris systems, which was a 25-fold increased secretion level of rhLK8 compared to the secretion level at the initiation of this study.

• 상하수도학회지
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• 제25권1호
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• pp.85-93
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• 2011

The structural components of microorganism are quite related to the toxin and environmental conditions. The vegetative and dormant cells are quite affected by the physical and chemical environments to survive and they will be dormant when they are in the extreme environment. The mechanism to activate the microorganisms however, is not well defined yet in the area of activation state and sporulation state through the analysis of EPS. Other than that even the main mechanism of prior to acquisition of analysis values is not well understood. Therefore, what kind of specific environment to affect the activation and sporulation will be discussed through the analysis of the extracellular polymeric substances(EPS). EPS are a high molecular weight mixture of polymers presenting both outside of cells and interior of microbial aggregates. They are a major complex materials in microbial aggregation for sustaining them together in a three dimensional matrix. Three commonly used extraction methods were applied to this study their effectiveness and quantification in extracting EPS from several Bacillus microorganisms and activated sludge. Three extraction methods used for this study are regular centrifugation with formaldehyde (RCF), Steaming, and EDTA extraction. The results of EPS contents such as the quantitative proteins, carbohydrates and the ratio of protein versus carbohydrate from the several species with the several specific methods showed in this research. This study aims to get comparable results of the quantitative production of EPS and the effectiveness of sedimentation for Bacillus microorganisms and activated sludge from several wastewater treatment plans. The results revealed that the protein amount extracted was the highest by the Steaming method of three extraction methods before sporulation and the carbohydrate amount extracted was the highest by the RCA method of three extraction methods after sporulation. The higher amount of protein compared with carbohydrate from Bacillus microorganisms affected higher sedimentation efficiency, however it could not be found the relation between the EPS production and sedimentation efficiency for the activated sludge.