• Journal of Dairy Science and Biotechnology
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• v.26 no.1
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• pp.5-10
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• 2008

This study was conducted to isolate protein components from culture supernatant of Lactobacillus casei. and measure anti-cancer activity. The protein components were isolated A and B on Ultrafiltration membrane(3, 10, 30, 100 KDa). And the protein components A and B were isolated fractions(number $3{\sim}9$) on FPLC. Experimental studies were progressed through the cell cytotoxicity and anti-cancer activities. Cell cytotoxicity test using human kidney normal cell(293) showed cytotoxicity of below 20% by the protein components A and B($100{\mu}g/mL$). The anti-cancer activity was increased up to 70% by the protein components A and B($100{\mu}g/mL$) in AGS(stomach cancer), A549(lung cancer), MCF-7 (breast cancer), SK-OV-3(ovary cancer) and LoVo(colon cancer). Cell cytotoxicity test was showed cytotoxicity of about 50% by the fractions(number 3, 8, 9) isolated FPLC. The others have not the cytotoxicity about the human normal cell. The anti-cancer activity was increased up to 70% by the fraction number 7 in cancer cell line. Therefore the components isolated from culture supernatant of Lactobacillus casei were showed anti-cancer activity.

• International Journal of Industrial Entomology and Biomaterials
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• v.2 no.1
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• pp.55-59
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• 2001

The vitellin of the red flour beetled Tribolium castaneum Herbst was purified and characterized. The vitellin of T. castaneum was purified by the FPLC techniques, anion exchange chromatography and gel permeation chromatography. In native-polyacrylamide gel electrophoresis, vitellin of T. castaneum was detected as a single band. This native vitellin has molecular weight of 440 kDa. The vitellin of T. castaneum is composed of three polypeptides, designated Vnl (178 kDa), Vn2 (168 kDa) and Vn3 (52 kDa) in SDS-polyacrylamide gel electrophoresis. Three subunits of vitellin were presented in the female adult hemolymph and egg extracts, but not observed in the male. These three polypeptides gradually decreased during embryogenesis. Polyclonal antiserum raised against purified vitellin reacted with the three polypeptides, Vnl, Vn2 and Vn3. Antisera raised against Vn1 and Vn2 cross-reacted with the two large subunits, Vnl and Vn2, respectively. Another subunits Vn3, however, was not cross-reacted with these two antisera. Also, antiserum raised against Vn3 did not cross-react with the Vn1 and Vn2.

• Microbiology and Biotechnology Letters
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• v.20 no.4
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• pp.401-408
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• 1992

The extracellular cholesterol oxidase produced from a soil microorganism HSL613 was purified and partially characterized. Through a series of purification procedures including concentration with CH2 concentrator, DEAE-cellulose column chromatography and gel filtration on Superose12, the purified enzyme was shown to have a specific activity of 108 units/mg protein giving 30.8-fold purification and final yield of 66%. The molecular weight of the enzyme was estimated to be 59,500 daltons by SDS-PAGE. The optimum temperature and pH for this enzyme were $50^{\circ}C$ and 6.0, respectively. The activity of the purified cholesterol oxidase was inhibited by $Ag^{2+}$, $Hg^{2+}$ and SDS.

• Biomolecules & Therapeutics
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• v.4 no.1
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• pp.46-50
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• 1996

Inhibition of the growth of human cancer cells by proteins secreted from 373-L1 cells was investigated in the present study. The growth of human cancer cells was inhibited by co-culture with 373-L1 cells under 10% FBS and DME, DME, GIT and serumless medium, respectively. The conditioned medium of cultured 373-L1 cells under serumless medium was concentrated 100-fold through an ultrafiltration cell with a 10,000 molecular weight cutoff at 4$^{\circ}C$ under positive pressure using nitrogen(373-L1 EM). 373-L1 EM inhibited the growth of HeLa, Hep G 2, KHOS-Np, A43l and MCF-7 cells. 3T3-L1 EM was purified with FPLC, DEAE-ion exchange chromatography and phenyl-sepharose chromatography. The major protein of 373-L1 EM has a molecular weight of 66,000-68,000 in SDS-PAGE analysis. The results suggest that the inhibitory activity of 373-L1 EM appears to be due to some protein(m.w.66,000-68,000) secreted by 373-L1 cells.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.228-234
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• 2002

Bacillus subtilis CAU131 (KCCM 10257) isolated from a fermented shrimp product produces subtilein, tentatively named as a bacteriocin, which exhibited a bactericidal effect against closely related species such as Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778, and several other strains of Bacillus sp. The purification of the subtilein was achieved by applying a mono-Q anion exchange chromatography on FPLC and $C_18$ reverse-phase chromatography on HPLC. After purification, specific activity of subtilein was increased about 3,000-fold compared with culture broth and its molecular mass was about 5,000 Da on SDS-PAGE. The antimicrobial activity of subtilein was well maintained at acidic and neutral pHs between 3 and 8. Subtilein was relatively heat stable, and its antimicrobial activity remained for 2 h at $80^{\circ}C$. However, the activity was reduced after heating at $100^{\circ}C$, and about $80\%$ of the activity was found after 1 h incubation at $100^{\circ}C$. The treatment of Bacillus subtilis ATCC 6633 with subtilein led to morphological changes in stationary-phase cells and most cells appeared to be lysed.

• BMB Reports
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• v.31 no.3
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• pp.274-280
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• 1998

An inositol monophosphatase (IMPase) was purified to homogeneity from rat duodenal mucosa for the first time and its enzymatic properties were investigated. Rat duodenal mucosa peculiarly exhibited the highest IMPase activity among various rat tissues examined. By means of ammonium sulfate precipitation, followed by Q-Sepharose, polylysine agarose, reactive-red agarose column chromatography, Uno-Q FPLC, and Bio-Silect FPLC, duodenal IMPase was purified 223-fold to a specific activity of 13.6 U/mg protein. The molecular mass of the native enzyme was estimated to be 48,000 Da on gel filtration. The subunit molecular mass was determined by SDS-PAGE to be 24,000 Da. These results indicate that duodenal IMPase is a dime ric protein made up of identical subunits. Rat duodenal IMPase has distinct properties from brain IMPase. It has a broad spectrum of substrate specificity and is insensitive to $Li^+$. Duodenal IMPase does not absolutely require $Mg^{2+}$ for its catalytic activity. Furthermore, duodenal IMPase is less stable to heat than brain enzyme. It is suggested that the rat duodenal mucosa needs a large amount of IMPase whose properties are quite different from that of the brain enzyme.

• BMB Reports
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• v.32 no.3
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• pp.239-246
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• 1999

A tripeptidase (PepT) was purified to homogeneity from Bacillus subtilis through four sequential chromatographies including DEAE-Sepharose ion exchange, hydroxylapatite, mono-Q FPLC ion exchange, and Superose-12 FPLC gel filtration. The apparent molecular mass of the enzyme was 49,200 Da and 51,400 Da as determined by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography, respectively, and the enzyme exists in a monomeric form. The physicochemical properties of the enzyme were as follows: optimum pH at 7.5, optimum temperature at $60^{\circ}C$, and pI at 4.9. The $K_m$ and $V_{max}$ values of the enzyme were 4.3 mM and 2.5 mmol/min/mg, respectively, with MetAla-Ser as substrate. The B. subtilis PepT requires $Co^{2+}$ ion(s) for activation, while it is inactivated by EOTA and 1,10-phenanthroline, suggesting that it is a metalloprotein. The enzyme was not inhibited by any of serine protease, aspartic protease, or leucine aminopeptidase inhibitors. The enzyme showed comparable activities towards four different substrates including Met-Ala-Ser, Leu-Gly-Gly, Leu-Ser-Phe, and Leu-Leu-Tyr. The amino terminal sequence of PepT determined by Edman degradation was found to be MKEEIIERFTTYVXV and turned out to be identical to that of PepT deduced from a cloned B. subtilis pepT.

• Journal of Life Science
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• v.8 no.5
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• pp.497-503
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• 1998

Cyclodextrinase from B. stearothemophilus KJ16 that can produce both cyclodextrin(CD) glucanotransferase and cyclodextrinase was purified 87.6-fold with 7% yield by ammonium sulfate precipitation, DEAE-cellulose chromatog-raphy, Sephadex G-100 chromatography, and FPLC. The molecular weight of the purified enzyme was about 68,000 dalton by SDS-PAGE. The optimal pH and temperature were 6.0 and 55$^{\circ}C$, respectively. The enzyme was stable at 5$0^{\circ}C$ for 2 hr in the pH range of 5.5 and 8.5. The enzyme activity was inhibited strongly by mercaptoethanol, di-thiothreitol, p-chloromercuribenzoate, N-bromosuccinimide, $Cu^{+2}$and $Hg^{+2}$. The purified enzyme hydrolyzed CDs with$\gamma$-CD>$\beta$-CD>$\alpha$-CD. The enzyme also hydrolyzed linear maltodextrins and polysaccharides, but the rates of hyd-rolysis for such substrates were slow as compared to that for $\gamma$-CD. The final degradation products with all substrates were maltose and glucose. Maltose was not further hydrolyzed.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.480-483
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• 2003

Fusion ferritin$(F_H+F_L)$, an iron-binding protein, was purified from recombinant E. coli by two-step sonications with urea. Unfolded ferritin was refolded by gel filtration chromatography with various concentration of urea. 50 mM Tris-HCl(pH 8.0) buffers with 1 M to 4 M urea were used in GFC. Objective was to characterize the structure change with urea concentration. Molecular weight was determined using GF-HPLC and RP-HPLC was used to quantify the unfolded and refolded proteins.

• Korean Journal of Microbiology
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• v.49 no.1
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• pp.95-98
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• 2013

Following construction of expression vectors in Escherichia coli, a new procedure involving two-step column purifications with a Fast Performance Liquid Chromatography System was developed for purification of the oxygenase component of alkylphenol hydroxylase of Pseudomonas alkylphenolia. From 50 g wet cake of recombinant E. coli BL21(DE3)(pJJPMO2) cells, 110 mg of pure protein in a heterodimeric form containing a stoichiometric amount of iron were obtained and it exhibited a specific activity of 147 nmole/min/mg.