• BMB Reports
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• v.28 no.2
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• pp.129-137
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• 1995

The ryanodine-receptor $Ca^{2+}$ release channel protein in the sarcoplasmic reticulum membrane of rabbit skeletal muscle plays an important role in muscle exitation-contraction (E-C) coupling. Various types of detergents were tested, including Chaps, cholate, octylglucoside, Zwittergents, Mega-9, Lubrol PX, and Triton X-100 for solubilization of this protein. Among these, Chaps and Triton X-100 were found to optionally solubilize the channel complex. Optimum conditions for this solubilization were pH 7.4 with a salt concentration of 1 M. The addition of phospholipid in the solubilization step helped in stabilizing the protein. The purification of the receptor was performed using sucrose density gradient centrifugation. Various methods [dilution, freeze-thaw, adsorption (Biobeads), and dialysis] were investigated to incorporate the Chaps-solubilized and purified $Ca^{2+}$ release channel protein into liposomes made from different types of phospholipids. Of these, a combined method consisting of a dialysis, freeze-thaw and sonication steps yielded the best results. Reconstituted vesicles produced by this method with 95% phosphatidylcholine (from soybean extract) had good function.

• Journal of Microbiology and Biotechnology
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• v.21 no.2
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• pp.129-135
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• 2011

Cloning and characterization of the gene encoding a solvent-tolerant protease from the haloalkaliphilic bacterium Geomicrobium sp. EMB2 are described. Primers designed based on the N-terminal amino acid sequence of the purified EMB2 protease helped in the amplification of a 1,505-bp open reading frame that had a coding potential of a 42.7-kDa polypeptide. The deduced EMB2 protein contained a 35.4-kDa mature protein of 311 residues, with a high proportion of acidic amino acid residues. Phylogenetic analysis placed the EMB2 gene close to a known serine protease from Bacillus clausii KSM-K16. Primary sequence analysis indicated a hydrophobic inclination of the protein; and the 3D structure modeling elucidated a relatively higher percentage of small (glycine, alanine, and valine) and borderline (serine and threonine) hydrophobic residues on its surface. The structure analysis also highlighted enrichment of acidic residues at the cost of basic residues. The study indicated that solvent and salt stabilities in Geomicrobium sp. protease may be accorded to different structural features; that is, the presence of a number of small hydrophobic amino acid residues on the surface and a higher content of acidic amino acid residues, respectively.

• Journal of Microbiology and Biotechnology
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• v.27 no.1
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• pp.84-91
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• 2017

The concern over the use of melittin in honey bee venom due to its adverse reaction caused by allergens such as phospholipase A2 ($PLA_2$) and hyaluronidase (HYA) has been an obstacle towards its usage. We developed a novel single-step method for melittin purification and the removal of $PLA_2$ and HYA. This study explores the influence of pH, buffer compositions, salt concentration, and types of cation-exchange chromatography resins on the recovery of melittin and the removal of both HYA and $PLA_2$. Melittin was readily purified with a strong cation-exchange resin at pH 6.0 with sodium phosphate buffer. It resulted in a recovery yield of melittin up to 93% (5.87 mg from a total of 6.32 mg of initial melittin in crude bee venom), which is higher than any previously reported studies on melittin purification. $PLA_2$ (99%) and HYA (96%) were also successfully removed. Our study generates a single-step purification method for melittin with a high removal rate of $PLA_2$ and HYA, enabling melittin to be fully utilized for its therapeutic purposes.

• Journal of Life Science
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• v.13 no.5
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• pp.718-722
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• 2003

Various marine microorganisms were isolated from seaweed, and their alginate-degrading activities were investigated. An alginate-degrading bacteria, Vibrio sp. AEBL-211, showed highest level of alginase activity when cultured on a mineral salt medium containing 0.7%(w/v) sodium alginate as the sole carbon source. The intracellular alginase from AEBL-211 was partially purified by ion chromatography on DE 52-cellulose column and gel filteration on Sepacryl G-200 column, and showed guluronate-specific 1yase activity.

• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.890-894
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• 2001

The isolation of genomic DNA from mammalian cells is usually performed by cell lysis followed by protein digestion, extraction, and finally, ethanol precipitation of the chromosomal DNA. However, in the case of large sample numbers or when only small amounts of starting materials are available, such conventional methods are not efficient and are cumbersome to be applied. Some alternative methods have been described as well as having commercial DNA isolation kits to be available, nevertheless, there is room left for much improvement. In the present study, a novel method is introduced, where it simplifies conventional protocols by omitting some time-consuming steps such as protease incubation or DNA precipitation and its resuspension. Using paramagnetic silica resins, the genomic DNA was purified over a magnetic field, and the bound DNA was eluted with a low-salt buffer. The fidelity and effectiveness of this novel method was determined by using normal and apoptotic cells as a starting material and then compared to other protocols. The high speed and convenience along with its high efficiency in detecting apoptotic chromosomal DNA will prove this method to be an improved alternative in the isolation of genomic DNA from mammalian cells.

• Journal of the Korean Society of Food Science and Nutrition
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• v.24 no.4
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• pp.636-641
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• 1995

Proteolytic activities were compared using three species involving in squid jeotkal fermentation and showing positive reaction upon casein test : Pseudomonas D2, Flavovacterium odoratum and Acinetobacter calcoaceticus. Pseudomonas D2 produced highest activity of protease at 72h when incubated in our own modified medium(polypeptone, 0.5% ; tryptone, 0.5% ; NaCl, 3% ; pH, 7.5). Thus, this specie was selected for the further study. The growth pattern was coincided with the production of protease. Thus purification of protease was proceeded by ethanol precipitation, sephadex G-100 gel filtration, and DEAE sepharose ion exchange chromatography. The purified protease showed highest activity at pH 7.0 and 5$0^{\circ}C$. The enzyme was very stable over the wide ragnes of the temperature ; even with one hour heat treatment at 7$0^{\circ}C$, the enzyme showed substantial amount of the activity toward casein. In addition, the enzyme was stable over the wide range of pH. Molecular weight of the protease was determined to be 17.4 kD by SDS-PAGE.

• Archives of Pharmacal Research
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• v.18 no.5
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• pp.308-313
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• 1995

This study has been undertaken to examine the acetylcholinesterase (AChE) of electric organ from korean electtric ray(Narke japonica). Korean electric ray was caughted at Chungmu sea and transported to the laboratory, where electric organs were removed and stored at $-70^{\circ}C$ until used. Acelycholinesterase(AChE) of electric organ was purified by affinity column that was prepared with dicaproyl-methylpyridinium linked to Sepharose 4B. Upon purification, the specific activities in Ellman unit were increased by 52 and 39 times for high salt soluble AChE (HSSE, 870.86 $\DeltaOD/min/geam$ of tissue) and detergent soluble AChE(DSE, 105.42 .$\DeltaOD/min/geam$ of tissue), respectively. Each subunit of AChE separated by SDS polyacrylamide gel electrophoresis(SDS-PAGE)was transferred to immonilon P by western boltting and detected by mAbs raised against each subunit of AChE from electric organ og Torpedo califomica. Collagenic tails of AChE from Torpedo califomica, likewise 103Kd protein of AChE from Narke japonica was detected by monoclonal antibody specific to 103Kd of AChE from Torpedo califomica. However, molar ratio of three subunits of AChE from Narke japonica is different from that of Torpedo calicormica. Furthermore, catalytic subunit of AChE from Narke japonica was not identified by monoclnal antibody specific to catalytic subunit of AChE from Torpedo californica. These results showed differences in molecular structure of AChE from Narke japonica and AChE from Torpedo califormica eventhough they showed same enzymatic activities.

• Microbiology and Biotechnology Letters
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• v.21 no.1
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• pp.54-58
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• 1993

During the screening of inhibitors against protein kinase CCPKC) and the bleb formation of K562 cell induced by phorbol ester from microbial secondary metabolites, MT-2007 was purified by solvent extraction, and chromatographic techniques from Actinomycetes isolate No. 2007-18. It showed completely suppression of bleb formation of K562 cell surface induced by phorbol 12.13dibutylate at the concentration of 503.9 11M and ICso on PKC was 31.4 11M. Its structure was postulated as lasalocid A sodium salt by physico-chemical properties and UV, IR. MS, IH-NMR.

• Clinical and Experimental Reproductive Medicine
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• v.17 no.2
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• pp.197-202
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• 1990

Early studies demonstrated that seminal plasma has a factor which inhibits fertilizing ability in a reversible manner. The factor can be precipitated by centrifugation at 104000g for 18 hr. The precipitate was applied to a CM cellulose column and eluted with high salt concentration. This fraction possessed antifertilizing activity was applied to a Sephacryl S-200 column according to a modification of the method of Reddy et al. Using such inhibition of in vitro fertilization ability as an assay, we have carried our experiments to purified the factor. When the factor was added to IVF medium, 70-80% of fertilization was inhibited.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.42 no.2
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• pp.134-140
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• 1997

The monoclonal antibody against a novel protein, which was induced in the roots of rice seedlings treated with NaCl, was produced. Oryzea sativa L. cv. Annapruna grains were sown on clean sands and grown for 10days and then the seedlings were soaked in 50mM NaCl aqueous solution. In 48hrs of the NaCl treatment, the roots were collected and homogenated with liquid nitrogen and extraction buffer. The homogenate was centrifuged and to the supernatant 75％ ammonium sulfate was added to pre-cipitate proteins. From these proteins a novel protein was purified through DEAE-ion chromatography and FPLC(Phenyl column). This protein appeared as a single band in the native electrophoresis. Using this protein as antigen, monoclonal antibody was produced. Five cell lines that secreted antibodies specifically bound to this protein were constructed.