• Journal of Microbiology and Biotechnology
• /
• v.25 no.7
• /
• pp.963-977
• /
• 2015

The well-characterized gram-positive bacterium Bacillus subtilis is an outstanding industrial candidate for protein expression owing to its single membrane and high capacity of secretion, simplifying the downstream processing of secretory proteins. During the last few years, there has been continuous progress in the illustration of secretion mechanisms and application of this robust host in various fields of life science, such as enzyme production, feed additives, and food and pharmaceutical industries. Here, we review the developments of Bacillus subtilis as a highly promising expression system illuminating strong chemical- and temperatureinducible and other types of promoters, strategies for ribosome-binding-site utilization, and the novel approach of signal peptide selection. Furthermore, we outline the main steps of the Sec pathway and the relevant elements as well as their interactions. In addition, we introduce the latest discoveries of Tat-related complex structures and functions and the countless applications of this full-folded protein secretion pathway. This review also lists some of the current understandings of ATP-binding cassette transporters. According to the extensive knowledge on the genetic modification strategies and molecular biology of Bacillus subtilis, we propose some suggestions and strategies for improving the yield of intended productions. We expect this to promote striking future developments in the optimization and application of this bacterium.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.8 no.3
• /
• pp.192-198
• /
• 2003

BiP, immunoglobulin binding protein, is an ER homologue of Hsp70. However, unlit other Hsp70 proteins, regulatory protein(s) for BiP has not been identified. Here, we demo strafed the presence of potential regulatory proteins for BiP using a pull -down assay. Since BiP can bind any unfolded protein, only the ATPase domain of BiP was used for the pull -down assay in order to minimize nonspecific binding. The ATPase domain was cloned to produce recombinant protein, which was then conjugated to CNBr-activated agarose. The structural conformation and ATP hydrolysis activity of the recombinant ATPase domain were similar to those of the native protein, light proteins from metabolically labeled mouse plasmacytoma cells specifically bound to the recombinant ATPase protein. The binding of these proteins was inhibited by excess amounts of free ATPase protein, and was dependent on the presence of ATP. These proteins were eluted by ADP. Of these proteins, Grp170 and BiP where identified. while the other were not identified as known ER proteins, from Western blot analyses. The presence of the ATPase-binding proteins for BiP was first demonstrated in this study, and our data suggest similar regulatory machinery for BiP may exist in the ER, as found in prokaryotes and other cellular compartments.

• Journal of the Korean Applied Science and Technology
• /
• v.25 no.3
• /
• pp.380-387
• /
• 2008

The effects of the applied stretch and MgADP binding on the structure of the actin and myosin cross-bridges in rabbit fibers in the rigor state have been investigatedwith improved resolution by x-ray diffraction using synchrotron radiation. To clarify the structure of the ATP hydrolysis intermediates formed by actin and myosin cross-bridges,the effects of various phosphate analogs in the of MgADP on the structure of the thin and thick filaments in glycerinated rabbit muscle fibers in the rigor state investigated by x-ray diffraction with a short exposure time using synchrotron radiation. These results strongly suggest that when MgADP and phosphate analogs such as metallofluorides(BeF3 and AlF4)and vanadate(VO4(Vi)) were added the rigor fibers in the presence of the ATP-depletion backup system, the intensities of the actin-based layer lines were markedly weakened. We found that the intensity of the 14.5 nm-based meridional reflections increase by 20-50% when phosphate analogs such as metallofluorides(BeF3 and AlF4) and vanadate(VO4(Vi)) was added to the rigor muscle.

• Korean Journal of Microbiology
• /
• v.43 no.4
• /
• pp.250-255
• /
• 2007

The RecA protein of Deinococcus radiodurans is essential for the extreme radiation resistance of this organism. The central steps involved in recombinational DNA repair require DNA-dependent ATP hydrolysis by recA protein. Key feature of RecA protein-mediated activities is the interactions with ssDNA and dsDNA. The ssDNA is the site where RecA protein filament formation nucleates and where initiation of DNA strand exchange takes place. The effect of sequence heterogeneity of ssDNA was examined in this experiment. The rate of homopolymeric synthetic ssDNA-dependent ATP hydrolysis was constant or nearly so over a broader range of pHs. For poly(dT)-dependent ATP or dATP hydrolysis, rates were generally faster, with a broader optimum between pH 7.0 and 8.0. Activities of RecA protein were affected by the ionic environment. The ATPase activity was shown to have different sensitivity to anionic species. The presence of glutamate seemed to slimulate the hydrolytic activity. Dr RecA protein was shown to require $Mg^{2+}$ ion greater than 2 mM for binding to etheno ssDNA and the binding stoichiometry of 3 nucleotide for RecA protein monomer.

• Proceedings of the PSK Conference
• /
• 2003.10b
• /
• pp.160.1-160.1
• /
• 2003

In order to confirm the biological function of ORF10 in cephabacin biosynthesis gene cluster of Lysobacter lactamgenus as an ATP-binding-cassette (ABC) transporter, the gene for ORF10 was amplified and subcloned into pET-28a(+) expression vector. After gene induction with 0.5 mM IPTG at 30~! and further cultivation at $30^~$ !. for 8 hr, a lot of the recombinant ORF10 protein was produced as soluble form in cytoplasmic fraction as well as a membrane protein in the membrane fraction as likely as other ABC transporters. (omitted)

• Korean Journal of Veterinary Research
• /
• v.40 no.2
• /
• pp.275-282
• /
• 2000

Substantia nigra is known to highly express glibenclamide binding site, a protein associated to ATP-sensitive $K^{+}$ ($K_{ATP}$) channel in the brain. However, the functional expression of $K_{ATP}$ channels in the area is not yet known. In this work, we attempted to estimate the functional expression of $K_{ATP}$ channels in neurons of the substantia nigra pars compacta (SNC) in young rats using slice patch clamp technique. Membrane properties and whole cell currents attributable to $K_{ATP}$ channel were examined by the current and voltage clamp method, respectively. In SNC, two sub-populations of neurons were identified. Type I (rhythmic) neurons had low frequency rebound action potentials ($4.5{\pm}0.25Hz$, n=75) with rhythmic pattern. Type II (phasic) neurons were characterized by faster firing ($22.7{\pm}3.16Hz$, n=12). Both time constants and membrane capacitance in rhythmic neurons ($34.0{\pm}1.27$ ms, $270.0{\pm}11.83$ pF) and phasic neurons ($23.7{\pm}4.16$ ms, $184{\pm}35.2$ pF) were also significantly different. The current density of $K_{ATP}$ channels was $6.1{\pm}1.47$ pA/pF (2.44~15.43 pA/pF, n=8) at rhythmic neurons of young rats. Our data show that in SNC there are two types of neurons with different electrical properties and the density of $K_{ATP}$, channel of rhythmic neuron is about 600 channels per neuron.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.1
• /
• pp.66-71
• /
• 2016

PikD is a widely known pathway-specific regulator for controlling pikromycin production in Streptomyces venezuelae ATCC 15439, which is a representative of the large ATP-binding regulator of the LuxR family (LAL) in Streptomyces sp. RapH and FkbN also belong to the LAL family of transcriptional regulators, which show greatest homology with the ATP-binding motif and helix-turn-helix DNA-binding motif of PikD. Overexpression of pikD and heterologous expression of rapH and fkbN led to enhanced production of pikromycin by approximately 1.8-, 1.6-, and 1.6-fold in S. venezuelae, respectively. Cross-complementation of rapH and fkbN in the pikD deletion mutant (ΔpikD) restored pikromycin and derived macrolactone production. Overall, these results show that heterologous expression of rapH and fkbN leads to the overproduction of pikromycin and its congeners from the pikromycin biosynthetic pathway in S. venezuelae, and they have the same functionality as the pathwayspecific transcriptional activator for the pikromycin biosynthetic pathway in the ΔpikD strain. These results also show extensive "cross-communication" between pathway-specific regulators of streptomycetes and suggest revision of the current paradigm for pathwayspecific versus global regulation of secondary metabolism in Streptomyces species.

• The Korean Journal of Zoology
• /
• v.13 no.3
• /
• pp.85-93
• /
• 1970

Measurements were made of the $Ca^++$ uptake, oxygen consumption and ATPase activity of mitochondria extracted from the rat liver in sucrose-tris chloride medium. $Ca^++$ binding of mitochondria was not affected by the incubation temperature in the range of $0^\\circ - 37^\\circ C$. Succinate did not increase the amount of $Ca^++$ bound while it increased oxygen consumption highly. The presence of ATP in the incubation medium did not enhance the $Ca^++$ uptake either. Therefore, it is concluded that the initial binding of $Ca^++$ is independent on metabolism.

• Journal of Pharmaceutical Investigation
• /
• v.36 no.5
• /
• pp.315-320
• /
• 2006

Silymarin showed P-glycoprptein(P-gp) inhibitory activity as much as verapamil, a well-known P-gp inhibitor, by decreasing $IC_{50}$ value of daunomycin(DNM)($16.0{\pm}0.7{\mu}M$), increasing the DNM accumulation($224.9{\pm}3.2%$), and decreasing DNM efflux($58.5{\pm}6.7%$), concurrently. In this study, we clarified the mechanism of action of silymarin for P-gp inhibitory function. First, silymarin may bind to the ATP-binding site and thus, prevent ATP hydrolysis. Second, the P-gp inhibitory activity of silymarin is not related to changing the cellular P-gp level. Third, the cytotoxicity of silymarin was increased in the presence of verapamil, reflecting that silymarin is a competent P-gp substrate against verapamil in the P-gp-overexpressed adriamycin-resistant MCF-7 breast cancer(MCF-7/ADR) cells. Conclusively, silymarin had the P-gp inhibitory activity through the action of competent binding to the P-gp substrate-binding site. Therefore, silymarin can be a good candidate for safe and effective MDR reversing agent in clinical chemotherapy by administering concomitantly with anticancer drugs.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.1
• /
• pp.15-19
• /
• 2006

A ferredoxin adsorbed hetero self-assembled layer was fabricated on chemically modified Au substrate, 4-Aminothiophenol (4-ATP) was deposited onto Au substrate and then N-succinimidyl-3-[2-pyridyldithio] propionate (SPDP) was adsorbed on the 4-ATP layer, since SPDP was used as a bridging molecule for ferredoxin adsorption, Ferredoxin/SPDP/4-ATP structured hetero layer was constructed because of strong chemical binding of ferredoxin, SPDP, and 4-ATP, The surface of the ferredoxin-adsorbed SPDP/4-ATP layer was observed by scanning tunneling microscopy, The hetero film formation was verified by surface plasmon resonance measurement. The current flow and rectifying property based on the scanning tunneling spectroscopy I-V characteristics was achieved in the proposed hetero layer. Thus, the hetero layer structure of ferredoxin functioned as a molecular diode with rectifying property, The proposed molecular diode can be usefully applied for the development of molecular scale electronic devices.