• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.635-638
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• 2004

An ATP-binding-cassette (ABC) transporter gene in the cephabacin biosynthetic gene cluster of Lysobacter lactamgenus was characterized. The amplified orf10 (cpbJ) gene was subcloned into pET-28a(+) vector and expressed in E. coli BL21(DE3) strain by 0.5 mM IPTG at $30^{\circ}C$. The membrane fraction of recombinant E. coli cells was separated by ultracentrifugation, and solubilized using 2.5% octyl-$\beta$-D-glucoside. Using the solubilized membrane fraction, the artificial proteoliposomes were reconstituted and analyzed for the biological activity of CpbJ protein. Upon measuring ATPase activity, the proteoliposome made from recombinant E. coli membrane proteins showed slightly higher activity than that from host E. coli membrane proteins. In the measurement of membrane transport activity, the reconstituted proteoliposome of recombinant E. coli membrane proteins exhibited higher activity when both substrates of cephalosporin C and L-Ala-L-Ser were applied, compared to the case of cephalosporin C or L-Ala-L-Ser only. It implies that the CpbJ protein is an ABC transporter secreting cephabacin antibiotics synthesized in cytoplasm.

• Journal of Microbiology and Biotechnology
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• v.3 no.3
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• pp.181-187
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• 1993

Steroid $\Delta^1$-dehydrogenase which introduces a double bond into the 1, 2 positions of steroid ring A was purified from Arthrobacter simplex, an excellent biotransformer of hydrocortisone into prednisolone. Hydrocortisone-induced cells were disrupted by vigorous agitation with glass beads, and a solubilized enzyme was obtained after centrifugation at 100, 000$\times$g for 90 minutes. The enzyme was purified 123-fold in three steps of chromatographic procedures with 13% yield. The last step of testosterone-agarose affinity column decisively contributed to the successful purification. The molecular weight of the enzyme was estimated to be 98, 000 by SDS-PAGE and 100, 000 by gel filtration, indicating that this enzyme behaves as a monomer. The enzyme showed demands for artificial electron acceptor, and among the several reagents tested, phenazine methosulfate acted as the most effective electron acceptor. Subcellular distribution of this enzyme was studied by centrifugation experiment. Comparison of the enzyme activities in pelleted membrane and cytosol fractions suggests that the enzyme may be a weakly attached peripheral membrane protein in vivo. But considerable amounts of enzyme was solubilized without any additional treatments for membrane protein.

• Korean Journal of Animal Reproduction
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• v.14 no.2
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• pp.93-100
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• 1990

These experiments were carried out to investigate the effect of rabbit anti-bovine cumulus cell antibodies on in vitro maturation of bovine follicular oocytes. Antisera to bovine cumulus cell were produced Japanese Ginat rabbit by repeated immunization of intact or solubilized bovine cumulus cell and purified by ammonium sulfate precipitation and Sepharose CL-4B protein-A affinity chromatography. The bovine cumulus cell-specific antibodies were confirmed by indirect ELISA. The results obtained in these experiments were summarized as follows : 1. The titer of the antibodies to cumulus cell determined by indirect ELISA using intact or solubilized bovine cumulus cell coated plates was very high in both intact and solubilized cumulus cells. Namely, the optical density at 1:12,800 dilution of antibodies was still significantly higher than that of non-immunized control serum. 2. When the follicular oocytes were treated with antibody to intact cumulus cells, the maturation rate of cumulus compacted and removed oocytes was ranged 47.6 to 59.1%. These value is significantly lower(p<0.05) than that(78.8%) of follicular oocytes cultured without the antibody. 3. the maturation rate of cumulus compacted and removed oocytes treated with antibody to solubilized cumulus cells was ranged 46.7 to 59.1%, significantly lower(p<0.05) than that(82.1%) of ooyctes cultured in antibody free medium. From above mentioned results, it could be said that cumulus cells promote nuclear maturation of follicular oocytes and that the beneficial effect of cumulus cells to the oocyte maturation is inhibited by the action of antibody to cumulus cells.

• Food Science and Preservation
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• v.8 no.2
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• pp.187-192
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• 2001

Amino acid composition of proteins in Italian millet, Common millet and sorghum were invstigated by HCI hydrolysis method. The optimum condition was obtained by hydrolysis at 110$\^{C}$ for 24hr. As major amino acids from protein hydrolyzate, the content of tyosine, arginine and phebylalanine were 7.06%, 6.79% and 6.44%, respectively. The content of glutamic acid in Common millet, Italian millet and Sorghum were 5.73%, 5.64% and 5.46%, respectively. Glycine content was about 2.93% in three samples. Contents of crude protein and pure protein in Italian millet, Common millet and sorghum were determined by micro-kjeldahl method. Crude protein contents were slightly higher than that of pure protein. Protein content of sorghum was higher than those of Italian millet and Common millet. For SDS-PAGE analysis, Italian millet showed more soluble proteins including 50kDa, 30kDa and smaller proteins than other cereals. In particular, Common millet and Sorghum only solubilized proteins less than 15kDa.

• Microbiology and Biotechnology Letters
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• v.26 no.4
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• pp.365-368
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• 1998

The aklavinone 11-hydroxylase which was overexpressed using T7 promoter in E. coli could be detected in SDS-PAGE only in insoluble precipitate without any detectable enzyme activity. The insoluble enzyme was solubilized in 6M guanidine$.$HCl solution and their refolding ability was tested under various conditions. When the enzymatic activity was checked by the bioconversion experiment, stepwise dialysis against 6M, 3M, 1M guanidine$.$HCl and finally 100 mM potassium phosphate buffer of the solubilized protein gave the best bioconversion efficiency. The aklavinone 11-hydroxylase showed its enzymatic activity in the reaction buffer containing NADPH with vigorous shaking. The enzymatic activity was lost during partial purification and regained by the addition of crude extract of S. lividans in the reaction mixture. This effect was confirmed to due to some low-molecular weight component(s) in the crude extract, because the addition of dialyzed crude extract could not recover the enzymatic activity.

• Journal of Microbiology and Biotechnology
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• v.3 no.4
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• pp.261-265
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• 1993

A sopA protein (41K) encoded by plasmid pXX288 was observed in the cytoplasm, whereas a sopB protein (37K) encoded by plasmid pXX157 was observed in the membrane fraction. Most of the sopB protein was solubilized from the crude membrane by treatment with Sarkosyl, which suggested that the protein may be located in the inner membrane. The sopA protein was precipitated at the concentration of 30 to 60% ammonium sulfate. The sedimentation profile of the crude membrane fraction showed a little difference according to culture media used, and the sopB protein existed in all fractions of inner membrane. The DNA of plasmids, pXX157, pXX300, and pXX167 co-sedimented with inner membrane fraction.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.500-504
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• 2005

Fusion ferritin (heavy chain ferritin, $F_H+$ light chain ferritin, $F_L$), an iron-binding protein, was primarily purified from recombinant Escherichia coli by two-step sonications with urea [1]. Unfolded ferritin was refolded by gel filtration chromatography (GFC) with refolding enhancer, where 50 mM Na-phosphate (pH 7.4) buffer containing additives such as Tween 20, PEG, and L-arginine was used. Ferritin is a multimeric protein that contains approximately 20 monomeric units for full activity. Fusion ferritin was expressed in the form of inclusion bodies (IBs). The IBs were initially solubilized in 4 M urea denaturant. The refolding process was then performed by decreasing the urea concentration on the GFC column to form protein multimers. The combination of the buffer-exchange effect of GFC and the refolding enhancers in refolding buffer resulted in an efficient route for producing properly folded fusion ferritin.

• Food Science and Biotechnology
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• v.14 no.1
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• pp.131-136
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• 2005

The selective extraction behavior of lactoferrin (Lf) from whey protein mixture was examined using reverse micelles formed by the cationic surfactant, cetyldimethylammonium bromide (CDAB). The major whey proteins, including ${\beta}$-lactoglobulin, ${\alpha}$-lactalbumin and bovine serum albumin, were solubilized from aqueous phase to organic phase while Lf was recovered in the aqueous phase. The solubilization behaviors of the proteins were manipulated by the process parameters such as the pH and salt concentration of the aqueous phase and the surfactant concentration in the organic phase. Efficient forward extraction was achieved with sodium borate buffer (50 mM, pH 9) containing 50 mM KCl and organic phase containing 100 mM CDAB. Based on SDS-PAGE and densitometry, about 96% of the initial Lf remained in the aqueous phase after forward extraction. The dialyzed Lf fully maintained its bacteriostatic activity against E. coli O157:H7.

• Proceedings of the Korean Biophysical Society Conference
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• 1996.07a
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• pp.33-33
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• 1996

An ectodomain of gp41, the transmembrane fusion protein of HIV, without the fusion peptide region was expressed using pET system in E. coli. The expressed protein gp41core, was isolated as inclusion body and was purified by ion-exchange chromatography after solubilized in 6M urea. The purified denatured protein was renaturated and the folded domain of gp41core was identified by the presence of the proteolysis resistence domain and a high content of ${\alpha}$-helical secondary structure. (omitted)

• KSBB Journal
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• v.18 no.1
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• pp.35-38
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• 2003

Analysis of proteome in amniotic fluid was performed by 2-D PAGE (polyacrylamide gel electrophoresis). Proteins in amniotic fluid were separated by centrifugation and solubilized in buffer solution for IEF, using an IPG strip of pH 4-7L. Both a homogeneous slab gel of 12.5% and a gradient gel of 8-18%, were used. After 2-D PAGE, spots were stained with silver nitrate and picked up for in-gel digestion. Digested peptides were analyzed by MALDI-TOF and proteins were further identifical. More protein spots were detected in the gradient gels and a protein not previously reported was identified.