• BMB Reports
• /
• 제40권5호
• /
• pp.723-730
• /
• 2007

Kinesin is an ATP-driven microtubule motor protein that plays important roles in control of microtubule dynamics, intracellular transport, cell division and signal transduction. The kinesin superfamily is composed of numerous members that are classified into 14 subfamilies. Animal kinesins have been well characterized. In contrast, plant kinesins have not yet to be characterized adequately. Here, a novel plant-specific kinesin gene, GhKCH2, has been cloned from cotton (Gossypium hirsutum) fibers and biochemically identified by prokaryotic expression, affinity purification, ATPase activity assay and microtubule-binding analysis. The putative motor domain of GhKCH2, $M_{396-734}$ corresponding to amino acids Q396-N734 was fused with 6$\times$His-tag, soluble-expressed in E. coli and affinity-purified in a large amount. The biochemical analysis demonstrated that the basal ATPase activity of $M_{396-734}$ is not activated by $Ca^{2+}$, but stimulated 30-fold max by microtubules. The enzymatic activation is microtubule-concentration-dependent, and the concentration of microtubules that corresponds to half-maximum activation was about 11 ${\mu}M$, much higher than that of other kinesins reported. The cosedimentation assay indicated that $M_{396-734}$ could bind to microtubules in vitro whenever the nucleotide AMP-PNP is present or absent. As a plant-specific microtubule-dependent kinesin with a lower microtubule-affinity and a nucleotide-independent microtubule-binding ability, cotton GhKCH2 might be involved in the function of microtubules during the deposition of cellulose microfibrils in fibers or the formation of cell wall.

• IMMUNE NETWORK
• /
• 제1권3호
• /
• pp.187-195
• /
• 2001

The expression of recombinant proteins fused to 26 kDa glutathione S-transferase (GST) extracted from Schistosoma japonicum represents an attractive system for purifiying proteins of interest in a single step using GST-affinity chromatography. In addition, the GST-tag is used conveniently for detecting fused proteins since its high solubility as well as its relatively small size rarely interferes with the biological activity of the fused protein. In this regard, the GST system is frequently applied for tracing fusion proteins in both prokaryotic and eukaryotic cells to elucidate the physiological interactions and functional compartments of proteins. To provide a further tool in analyzing GST-fusion proteins, a new monoclonal antibody, with a high specificity to the S. japonicum GST was produced. Methods: BALB/c mice were immunized both with recombinant S. japonicum GST proteins, and by the fusion of splenocytes from these mice with myeloma cells. From this, a new anti -GST monoclonal antibody, termed SARAH, was generated. The specificity and reactivity of this antibody was confirmed by ELISA and by Western blot analysis. Results: SARAH showed a high reactivity to recombinant GST and GST fusion protein but not with native mammalian GST proteins as derived from other species including humans, cows, rabbits and rats. The applicability of SARAH was further demonstrated by confocal laser scanning microscopy, where GST proteins that were expressed transiently in mouse fibroblast cells, were specifically detected without interference of endogenous GST. Conclusion: SARAH is new monoclonal antibody with a high specificity to recombinant GST proteins but not to endogenous GST in mammalian cells.

• BMB Reports
• /
• 제34권4호
• /
• pp.379-384
• /
• 2001

The cDNA encoding ribosomal protein was identified from a cDNA library of Schizosaccharomyces pombe. The nucleotide sequence of the 548 by cDNA clone reveals an open reading frame, which encodes a putative protein of 166 amino acids with a molecular mass of 18.3 kDa. The amino acid sequence of the S. pombe L11 protein is highly homologous with those of rat and fruit, while it is clearly less similar to those of prokaryotic counterparts. The 1,044 by upstream sequence, and the region encoding N-terminal 7 amino acids of the genomic DNA were fused into the promoterless $\beta$-galactosidase gene of the shuttle vector YEp357 in order to generate the fusion plasmid pHY L11. Synthesis of $\beta$-galactosidase from the fusion plasmid varied according to the growth curve. It decreased significantly in the growth-arrested yeast cells that were treated with aluminum chloride and mercuric chloride. However, it was enhanced by treatments with cadmium chloride ($2.5\;{\mu}M$), zinc chloride ($2.5\;{\mu}M$), and hydrogen peroxide (0.5 mM). This indicates that the expression of the L,11 gene could be induced by oxidative stress.

• Journal of Microbiology and Biotechnology
• /
• 제28권6호
• /
• pp.987-996
• /
• 2018

Bacterial programmed cell death is regulated by the toxin-antitoxin (TA) system. YhaV (toxin) and Pr1F (antitoxin) have been recently identified as a type II TA system in Escherichia coli. YhaV homologs have conserved active residues within the C-terminus, and to characterize the function of this region, we purified native YhaV protein (without denaturing) and constructed YhaV proteins of varying lengths. Here, we report a new low-temperature method of purifying native YhaV, which is notable given the existing challenges of purifying this highly toxic protein. The secondary structures and thermostability of the purified native protein were characterized and no significant structural destruction was observed, suggesting that the observed inhibition of cell growth in vivo was not the result of structural protein damage. However, it has been reported that excessive levels of protein expression may result in protein misfolding and changes in cell growth and mRNA stability. To exclude this possibility, we used an [$^{35}S$]-methionine prokaryotic cell-free protein synthesis system in vitro in the presence of purified YhaV, and two C-terminal truncated forms of this protein (YhaV-L and YhaV-S). Our results suggest that the YhaV C-terminal region is essential for mRNA interferase activity, and the W143 or H154 residues may play an analogous role to Y87 of RelE.

• Journal of Microbiology and Biotechnology
• /
• 제19권5호
• /
• pp.502-510
• /
• 2009

Although the Escherichia coli heat-labile enterotoxin B subunit (LTB) has already been expressed in several different systems, including prokaryotic and eukaryotic organisms, studies regarding the synthesis of LTB into oligomeric structures of pentameric size in the budding yeast Saccharomyces cerevisiae have been limited. Therefore, this study used a functional signal peptide of the amylase 1A protein from rice to direct the yeast-expressed LTB towards the endoplasmci reticulum to oligomerize with the expected pentameric size. The expression and assembly of the recombinant LTB were confirmed in both the cell-free extract and culture media of the recombinant strain using a Western blot analysis. The binding of the LTB pentamers to intestinal epithelial cell membrane glycolipid receptors was further verified using a GM1-ganglioside enzyme-linked inmmunosorbent assay (GM1-ELISA). On the basis of the GM1-ELISA results, pentameric LTB proteins comprised approximately 0.5-2.0% of the total soluble proteins, and the maximum quantity of secreted LTB was estimated to be 3 mg/l after a 3-day cultivation period. Consequently, the synthesis of LTB monomers and their assembly into biologically active aligomers in a recombinant S. cerevisiae strain demonstrated the feasibility of using a GRAS microorganism-based adjuvant, as well as the development of carriers against mucosal disease.

• KSBB Journal
• /
• 제22권5호
• /
• pp.356-361
• /
• 2007

시아노박테리아 같은 수중 미생물에 대한 2차대사물질에 대한 연구는 육상식물이나 미생물에 관련된 연구방법을 응용하고 있으며 아직까지 체계화 되어있지 않아 시아노박테리아를 보다 효율적으로 조사하기 위한 새로운 연구기술의 모색이 절실히 필요하다. 이 연구에서는 의학적인 관점에서 시아노박테리아를 조사하기 위한 유용한 접근 방법을 모색하였고 체계화시켰다. 균주마다 특성화된 최적 배양을 하였고 PCR 증폭을 이용한 분자생물학적인 방법과 HPLC를 통한 정성분석으로 의학적으로 의미있는 NPs를 생산하는 유망 균주를 선별하였다. 선별된 균주에서 나온 추출액은 SPE와 preparative HPLC를 거쳐 분리 정제되어지고, 정제된 물질들은 질량분석기에 의해 분자량과 구조가 결정되어 졌으며, 생활성 테스트에 의해서 그 생물학적인 활성을 정함으로써 의학적인 가능성이나 활용성에 대해서 고찰하였다. 이번 시험에서 98개의 실험균주 중 46개의 균주가 NRPS 또는 PKS 관련 gene을 함유하고 있었으나 HPLC를 이용한 정성분석에서 단지 5개의 균주가 상당히 의미있는 관련 단백질을 생산하고 있음이 알려졌다. 즉, 41 균주에서 관련 gene은 존재하였지만 그 발현이 미약하였고 또는 프로모터의 활성이 이루어지지 않았다. 이와같은 휴먼 gene의 활성화는 자외선 조사 또는 건조를 이용한 자극과 배양액의 성분 조절로 이루어질 수 있고 이를 통해 흥미있는 결과를 이끌어낼 수 있다(13, 14). 이번 연구에서는 HPLC를 통한 정성분석은 Biomass에 대해서 이루어졌다. 그러므로 배양액으로 유출되는 extracellular substances에 대한 분석은 행해지지 않았다. 시아노박테리아가 NRPS/PKS 관련 유전자를 함유하고 있을 때, 특히 biomass에서 NPs 이 검출되지 않을 경우 배양액 안에서의 존재 가능성 대해서 조사할 필요성이 요구되어진다. 시아노박테리아는 대표적인 photoautotroph 미생물로써 $CO_2$를 탄소성분과 에너지원으로 사용한다. 그러므로 E. coli 같은 미생물보다 배양시 경제적이고 또한 다른 종류의 미생물이 탄소영양분의 부족과 cyanobacterial NPs의 생물학적 환성에 의해서 공생하기 어려워 옥외배양을 통한 대량생산이 가능하다. 그러나, 생산되는 cyanobacterial NPs의 높은 구조적 안정성까지 포함하여 시아노박테리아는 미래의학산업의 중요한 천연소스임에 틀림이 없지만 개체 분화의 시간이 4$\sim$10시간 정도로 다른 미생물에 비해 길고 배양시 광원의 필요성 등 한계 요소를 지니고 있다(15). 그러므로 최근에는 개체 분화가 빠른 시아노박테리아나 또는 다른 미생물에 대해서 cyanobacterial NRPS/PKS gene의 heterologous expression 이 연구되어지고 있다(16).