• BMB Reports
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• v.40 no.5
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• pp.723-730
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• 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
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• v.1 no.3
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• pp.187-195
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• 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
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• v.34 no.4
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• pp.379-384
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• 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
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• v.28 no.6
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• pp.987-996
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• 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
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• v.19 no.5
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• pp.502-510
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• 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
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• v.22 no.5
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• pp.356-361
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• 2007

Cyanobacteria are a very old group of prokaryotic organisms that produce very diverse secondary metabolites, especially non-ribosomal peptide and polyketide structures. Although some cyanobacteria produce lethal toxins such as microcystins and anatoxins, some may be useful either for development into commercial drugs or as biochemical tools. Detection of unknown secondary metabolites was carried in the present study by a screening of 98 cyanobacterial strains from Cyanobiotech GmbH in order to establish a screening process, isolate pure substances and determine their bioactivities. A degenerated polymerase chain reaction technique as molecular approaches has been used for general screening of NRPS gene and PKS gene in cyanobacteria. A putative PKS gene was detected by DKF/DKR primer in 38 strains (38.8%) and PCR amplicons resulted from a presence of NRPS gene were showed by MTF2/MTR2 primer in 30 strains (30.6%), respectively. A screening of interesting strains was performed by comparing PCR screening results with HPLC analyses of extracts. HPLC analysis for a detection of natural products was performed in extracts from biomass. 5 strains were screened for further scale-up processing. 7 pure substances were isolated from the scale-up cultures and tested for bioactivities under consideration to purity, amount and molecular weight of substances. One substance isolated from CBT 635 showed cytotoxic activity. This substance may be regarded as Microcystin LR.