• Applied Biological Chemistry
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• v.26 no.1
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• pp.65-72
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• 1983

The composition of four rice protein groups is greatly affected by the extraction conditions. The extraction amounts of albumins and glubulines primarily depended on the temperature rather than the method of extraction. The total amount of glutelins, the major components of rice storage proteins, could be extracted by a successive extraction processes, extraction with 0.5% SDS-0.1M borate buffer(pH 8.3) followed by extraction with 0.5% SDS-0.6% ${\beta}-mercaptoethanol-0.1M$ borate buffer(pH 8.3). The extracted amounts of glutelin with these solvents were 54.1 and 45% respectively. The further purification of SDS soluble glutelins was achieved by Sephadex G-150 gel column chromatography. The molecular weight of the components in four protein groups has been estimated by SDS-polyacrylamide gel electrophoresis with or without ${\beta}-mercaptoethanol.$ The comparison of albumins and globulins by starch gel electrophoresis at pH 3.1 permitted us to identify seven rice varieties. However, at pH 8.95, the specific bands for Japonica type rice varieties were observed.

• Microbiology and Biotechnology Letters
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• v.34 no.1
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• pp.40-46
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• 2006

A bacterial strain producing high level of a phytase was isolated from cattle feces and identified as Bacillus subtilis, and designated as Bacillus sp. CF 5-26. The production of the phytase from Bacillus sp. CF 5-26 reached the highest level after 72 hours at $37^{\circ}C$. The optimum condition of the media for the production of phytase was 10% rice bran extract, 0.1% whey protein powder, $0.01%\;CaCl_{2},\;0.01%\;KH_{2}PO_4$. The phytase was purified 20.3 folds with ethanol precipitation, Sephadex G-100, CM Sepharose CL-6B and Sephacryl S-100-HR column chromatography. The molecular weight of the purified enzyme was estimated to be 66 kDa on SDS-polyacrylamide gel electrophoresis. The purified phytase activity was stable up pH 5.0, 7.0, 11.0 and the remaining activity was 50% when it was treated at $100^{\circ}C$ for 1 hour. The substrate specificity of phytase was most active against sodium phytate and inositol polyphosphate compound. And the phytase hydrolysed tripolyphosphate and pyrophosphate a little. The Km value for the sodium phytate was 0.64 mM and the Vmax value was $4.41\;{\mu}mol/min$.

• The Korean Journal of Food And Nutrition
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• v.17 no.1
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• pp.47-52
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• 2004

In this study, I attempted to develope the expression and purification system of human mammaglobin proteins in Escherichia coli and to produce anti-human mammaglobin rabbit antibody for the detection of human mammaglobin protein in the peripheral blood of breast cancer patients. Human mammaglobin gene was cloned and sequenced from m-RNAs purified from donated breast cancer tissues using RT-PCR. The cloned gene was inserted into pET30, pET22, and pET32 plasmid. The cloned gene in pET30 yields insoluble proteins which was difficult to purify from the cells extracts. The mammaglobin gene in pET32 was strongly expressed soluble proteins which were isolated using Ni-NTA affinity chromagraphy and DEAE-ion exchange chromatography, followed by enterokinase digestion of the purified proteins. The isolated proteins had enough purity to use as a antigen for the production of anti-mammaglobin antibody in rabbits. The polyclonal antibody produced against the isolated mammaglobin showed a specificity to mammaglobin after Westernblot immuno assay. In conclusion, the isolated mammaglobin protein and the anti-mammaglobin rabbit antibody may be used for diagnosis of breast cancer as well as development of anti-breast cancer drug.

• Korean Journal of Microbiology
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• v.39 no.3
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• pp.148-154
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• 2003

Moraxella sp. CK-l is known to inhibits the growth of Anabaena cylindrica, a cyanobacterium. It has been documented that the ability of this growth inhibition of Anabaena cylindrica was attributed to extracellular autolysin from Moraxella sp. CK-l. However, it remains to be elucidated identification and characterization of autolysin have yet been elucidated. In this study, we tried to purify and identify autolysin secreted from Moraxella sp. CK-l. Cells were grown in a complex liquid medium (BGC-11) and culture supernatants were collected, followed by ammonium sulfate fractionation. Fractions were further separated with anion exchange column, Mono-Q, in FPLC system and analyzed by SDS/PAGE. The fraction containing high autolysin activity showed a single distinct protein peak in anion column and molecular mass of about 17 kDa in SDS/PAGE. Nterminal amino acid sequencing of the protein was analyzed, of which result showed the homology with some proteases, including extracellular serine protease, Dichelobacter nodosus.

• Korean Journal of Microbiology
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• v.30 no.2
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• pp.101-107
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• 1992

Cytochrome c oxida5e from chemotrophically grown R p , geliitinosu was purified by cytochrome c affinity chromatography and DEAE-Sephacel ion exchange chromatography. The molecular weight of the cytochrome c oxidase was approximately 110.000 Da by sephacryl s-300 gel chromatography and approximately 52, 000 Da by SDS-gel electrophoresis, respectively. Therefore. cytochrolne c oxidase of Rps. gehtinosu seems to be dimer. The cytochrome c oxidasc was very sensitive to temperature. It's Km and Vmax were 20 pM and 44 unitlmg protein for horsc heart cytochrome c as a substrate. respectively, and its optimum pH and temperature were 6.4 and 25$^{\circ}$C. respectively. The absorption peaks of the reduced cytochrome c oxidase showed at 554 nm, 523 nm. and 422 nm. The activiiy of cytochrome c oxidase was inhibited by KCN, and NaN3, but not by CO, antimycir~ A. and myxothiazol. The cytochrome c-551 was produced either in phototrophically or chemotrophically grown Rps. gelaiinosci. The rcduced cytochrome c-551 was oxidized by b-type cytochrome c oxidase from Rp.v. gc.lrtino.sc~. Km and Vmax of cytochrome c oxidase was 26 pM and 31 unitlnlg protein For cytochrome c-551 as a substrate. respectively. Thercfore. thc electron transfer chain of chemotrophically grown Rps. glatinosa seems lo be ubiquinol cytochrome bc, complex -'cytochrome c-55lMb-type cytochrome c oxidase+02.

• Applied Biological Chemistry
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• v.37 no.1
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• pp.43-48
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• 1994

A basic inducible protein, ICG, containing chitinase and ${\beta}-1,3-glucanase$ activity concomittantly was purified from cell suspension culture media of rice after the treatment of chitooligosaccharides. The isolated ICG enzyme gave a single band on native and SDS polyacrylamide gel electrophoresis and its molecular weight was estimated to be 52.53 kd. The optimal temperature and optimal pH of both enzyme activities in ICG were $60^{\circ}C$, pH 6.0 for chitinase activity and $37^{\circ}C$, pH 4.0 for ${\beta}-1,3-glucanase$ activity. $K_M$ and $V_{max}$ values for chitinase were 0.474 mM. 2.997 nM/min., and those for ${\beta}-1,3-glucanase$ were 1.004 mM 0.739 nM/min. respectively. TLC analysis of the chitooligosaccharide hydrolysates with ICG enzyme indicated that ICG acts as endochitinase.

• Applied Biological Chemistry
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• v.36 no.2
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• pp.105-110
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• 1993

Inulin fructotransferase from Enterobacter sp. S45 was purified with DEAE-cellulose column chromatography and fast protein liquid chromatography. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight was estimated to be 42,800 by SDS-polyacrylamide gel electrophoresis. The optimal pH and temperature for the enzyme reaction were pH 5.5 and $55^{\circ}C$, respectively. $Mg^{2+}$ activated the enzyme activity, but $Fe^{3+}$, $Cu^{2+}$, $Hg^{2+}$ significantly inhibited. After exhaustive digestion of inulin by the enzyme, DFA III, sucrose, 1-kestose and nystose were produced. Sucrose, 1-kestose, raffinose and melezitose can't be used as substrates by the enzyme, but nystose and 1-F-fructofuranosyl nystose were hydrolysed. The Km and Vmax for inulin of the enzyme were 1.4 mM and $0.196\;{\mu}mole/min$, respectively.

• Korean Journal of Microbiology
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• v.44 no.3
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• pp.178-185
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• 2008

Avian influenza virus (AIV) is recognized as key to the emergence of pandemic influenza for humans; there are growing concerns that AIV H9N2 may become more efficient to transmit to humans in the near future, since the infection of poultry with AIV H9N2 has been common in recent years. In this study, we aimed to produce antisera recognizing the HA and NA proteins of AIV H9N2. Initially, coding sequences corresponding to the N-terminal regions of the HA and NA proteins of the Korean AIV H9N2 (A/Ck/Kr/MS96/96) isolated from a domestic chicken were amplified from the genomic RNA. Following cloning of the amplified cDNA fragments into pGEX4T-1 vector, two GST-fusion proteins (GST-HAln and GST-NAn) were expressed in E. coli BL21 and purified with glutathione sepharose columns; the recombinant GST-HAln and GST-NAn proteins were both used as immunogens in rabbits. The antigenicity of the rabbit antisera was analyzed by immunoblotting of the cell lysates prepared from AIV H9N2-infected MDCK cells. Overall, the recombinant HAln and NAn proteins fused to the C-terminus of GST and the rabbit antisera raised against the corresponding recombinant proteins would provide a valuable reagent for AIV diagnosis and basic research.

• BMB Reports
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• v.52 no.8
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• pp.496-501
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• 2019

Conventionally, immunotoxins have been produced as a single polypeptide from fused genes of an antibody fragment and a toxin. In this study, we adopted a unique approach of chemical conjugation of a toxin protein and an antibody fragment. The two genes were separately expressed in Escherichia coli and purified to high levels of purity. The two purified proteins were conjugated using a chemical linker. The advantage of this approach is its ability to overcome the problem of low recombinant immunotoxin production observed in some immunotoxins. Another advantage is that various combinations of immunotoxins can be prepared with fewer efforts, because the chemical conjugation of components is relatively simpler than the processes involved in cloning, expression, and purification of multiple immunotoxins. As a proof of concept, the scFv of trastuzumab and the PE24 fragment of Pseudomonas exotoxin A were separately produced using E. coli and then chemically crosslinked. The new immunotoxin was tested on four breast cancer cell lines variably expressing HER2. The chemically crosslinked immunotoxin exhibited cytotoxicity in proportion to the expression level of HER2. In conclusion, the present study revealed an alternative method of generating an immunotoxin that could effectively reduce the viability of HER2-expressing breast cancer cells. These results suggest the effectiveness of this method of immunotoxin crosslinking as a suitable alternative for producing immunotoxins.

• BMB Reports
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• v.36 no.6
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• pp.545-551
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• 2003

A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The $K_m$ and $V_{max}$ values for $NAD^+$ were 0.1 mM and $1.08\;{\mu}mol/min/mg$, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - $100\;{\mu}M$, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.