• Journal of Animal Science and Technology
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• v.47 no.6
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• pp.1025-1032
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• 2005

This study was carried out to separate ovotransferrin in chicken egg white by gel chromatography and heparin affinity chromatography. In gel filtration which was performed with 50mM Phosphate buffer (pH 7.2, 0.15M salt) at a flow rate of 2.0 ml/min, ovotransferrin and ovalbumin were eluted together in fraction number 11-16. In order to separate pure ovotransferrin, fraction No. 12-14 of them which have high concentration of ovotransferrin were concentrated and rechromatographed. However, the ovotransferrin did not separated clearly. In heparin affinity chromatography, the separation was performed with 50mM ethylaminetetraacetic acid (EDTA, pH7.2) and 50mM Phosphate buffer (pH 7.2, 0.15M salt contained) on ferrous and ferric ion saturated column at as same flow rate as gel filtration system's. Ovotransferrin and albumin were eluted together at 10-15min (fraction No.3) and 15-20min (fraction No.4), respectively. However, purified ovotransferrin was eluted at 156-165min and 165-175min (tube No.32-33) with 50 mM phosphate buffer (pH 7.2, 0.15M salt free), respectively. Heparin affinity chromatography with ferric ion saturated column was resulted in the best separation of ovotransferrin rather than separation by gel chromatography and ferrous ion saturated heparin affinity chromatography.

• Membrane Journal
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• v.19 no.2
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• pp.113-121
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• 2009

Porous affinity chitosan and chitin membranes with good mechanical strength and high protein binding capacity were prepared by using silica particles as porogen. The maximum binding capacity of affinity chitosan membrane for BSA protein is 21.8mg/mL, and that of affinity chitin membrane for lysozyme enzyme is 26.1mg/mL. Chromatographic separations of BSA and lysozyme proteins using the porous affinity chitosan and chitin membranes were performed with change of the flow rate, loading amount and concentration of protein loading solutions. Protein eluted amount and binding yield were calculated from the filtration chromatograms consisted of loading/washing/elution sequences. Protein binding amount and yield were increased with decreasing of flow rate, increasing of loading amount and concentration of protein loading solutions. Those results suggest that the porous chitosan and chitin membranes prepared by using silica particles as porogen are suitable in affinity filtration chromatography for large scale separation of proteins.

• YAKHAK HOEJI
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• v.41 no.5
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• pp.638-646
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• 1997

The steroid 9${alpha}$-hydroxylase activity has been detected in cytosol fraction, $100,00{\times}g$ supernatant of cell free extract of Mycobacterium fortuitum. The activity was not linear with protein concentration in the assay suggesting 9${alpha}$-hydroxylase is a multicomponent enzyme. The 9${alpha}$-hydroxylase system was partially purified through fractional saturation of ammonium sulfate, strong anion exchange (Mono Q) column chromatography, gel filtration (Superose 12) column chromatography, and testosterone affinity gel chromatography. Ammonium sulfate 50~60% saturated fraction of the cytosol gave 9${alpha}$-hydroxylase activity. For further purification, the half-saturated ammonium sulfate fraction was applied to Mono Q, Superose 12, or affinity gel column. The purification factors of 9${alpha}$-hydroxylase containing fraction after Mono Q, Superose 12, and affinity gel chromatography was 13, 11, and 17 respectively.

• BMB Reports
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• v.28 no.3
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• pp.197-203
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• 1995

Famesyl protein transferase involved in the first step of post-translational modification of $p21^{ras}$ proteins transfers the famesyl moiety from famesyl pyrophosphate to a cysteine residue in $p21^{ras}$ proteins. The enzyme was first purified 30,000-fold from bovine testis by use of 30~50% ammonium sulfate fractionation, DEAE-Sephacel ion exchange chromatography, Sephacryl S-300 gel filtration chromatography, Sephacryl S-200 gel filtration chromatography, and hexapeptide (Lys-Lys-Cys-Val-Ile-Met) affinity chromatography. The molecular weight of the purified enzyme was estimated to be ~100 kDa by gel filtration and SDS-polyacrylamide gels showed two closely spaced bands of ~50 kDa protein. These indicate that the enzyme consists of two nonidentical subunits, a and 13, which have slightly different molecular weights. The enzyme was inhibited by hexapeptide (Lys-Lys-Cys-Val-Ile-Met), which acted as an alternative substrate that competed for famesylation. Kinetic analysis by measuring initial velocities showed that famesyl protein transferase is a very slow enzyme. EDTA-treated famesyl protein transferase showed little activity with $Mg^{2+}$ or $Zn^{2+}$ alone, but required both $Mg^{2+}$ and $Zn^{2+}$ for the catalytic activity.

• Proceedings of the Membrane Society of Korea Conference
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• 2004.05a
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• pp.59-62
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• 2004

Affinity membranes have emerged principally to overcome the problems of limited specificity experienced with membranes that operate purely on a sieving mechanism and as an alternative to the traditional affinity resins. It is a logical expectation that affinity membranes might combine the outstanding selectivity of affinity resins with the high productivity associated with filtration membranes.(omitted)

• Membrane Journal
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• v.16 no.1
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• pp.39-50
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• 2006

Porous chitosan and chitin membranes were prepared by using silica particles as porogen. Membrane preparation was achieved via the following three steps: (1) chitosan film formation by casting an chitosan solution containing silica particles, (2) preparation of porous chitosan membrane by dissolving the silica particles by immersing the film into an alkaline solution and (3) preparation of porous chitin membrane by acetylation of chitosan membrane with acetic anhydride. The optimum preparation conditions which could provide a chitosan and chitin membranes with good mechanical strength and adequate pure water flux were determined. To allow protein affinity, a reactive dye (Cibacron Blue 3GA) was immobilized on porous chitosan membrane. Binding capacities of affinity chitosan and chitin membranes for protein and enzyme were determined by the batch adsorption experiments of BSA protein and lysozyme enzyme. The maximum binding capacity of affinity chitosan membrane for BSA protein is about 22 mg/mL, and that of affinity chitin membrane for lysozyme enzyme is about 26 mg/mL. Those binding capacities are about $several{\sim}several$ tens times larger than those of chitosan and chitin-based hydrogel beads. Those results suggest that the porous chitosan and chitin membranes are suitable in affinity filtration chromatography for large scale separation of proteins.

• Journal of Dairy Science and Biotechnology
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• v.23 no.1
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• pp.1-8
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• 2005

The purpose of this study was to demonstrate biochemical properties and antibacterial activity of lactoferrin(Lf) obtained from the colostrum of Korean native cow. Lactoferrin was isolated from the colostrum of Korean native cow by purification steps using batch extraction, ion exchange chromatography, gel filtration chromatography, affinity chromatography. Other whey protein components that is similar molecular weight and affinity to lactoferrin were gradually removed from crude Korean Native cow's lactoferrin during the purification steps. The molecular weight of the purified Korean native cow's Lf(K-Lf) was 81 kDa, the isoelectric point was 9, and the content of iron was 0.56mg/g, which is indicated that iron saturation of the K-Lf was 40.6%. Amino acid composition and a-helix content were different K-Lf from bovine Lf(B-Lf). Antibacterial activity of E. coli O111 by K-Lf was lower than that of B-Lf. A minimal inhibitory concentration(MIC) of K-Lf and B-Lf was 2.75mg/ml and 1.5mg/ml respectively.

• Applied Biological Chemistry
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• v.36 no.4
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• pp.255-259
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• 1993

Chitobiase from Aeromonas salmonicida YA7-625 was purified from culture broth through ammonium sulfate precipitation, chitin affinity adsorption, hydroxylapatite column chromatography and gel filtration, with 47.2% yield and 31.5 fold purity. The molecular weight of purified chitobiase was 15,000 daltons, and the chitobiase showes maximum activity at the condition of at $40^{\circ}C$ and pH 6.0. The effects of various metal ions and inhibitors show thatcystein, glutamic acid, serine, tryptophan, and tyrosine residues seem to be concerned in chitobiase activity.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.2
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• pp.144-149
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• 2001

For the production and purification of a single chain human insulin precursor, four types of fusion peptides $\beta$-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)(sub)6-tagged sequence (HTS) were investigated. Recombinant E. coli harboring hybrid genes was cultivated at 37$\^{C}$ for 1h, and gene induction occurred when 0.2mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except for E. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0mM IPTG, followed by a longer than 4h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique.

• Korean Journal of Microbiology
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• v.32 no.4
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• pp.315-321
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• 1994

Glyoxalase I was purified 2,294-fold from Pleurotus ostreatus by S-hexylglutathione affinity chromatography, Sephadex G-150 gel filtration chromatography and DEAE-sepharose A-50 CL-6B ion exchange chromatography with an overall yield of 21.7%. The molecular mass determined by gel filtration was found to be approx. 34 kDa. SDS-PAGE revealed that the enzyme consists of two identical subunits with a molecular mass of approx. 17 kDa. The K sub(m) values of this enzyme for methylglyoxal and phenylglyoxal were 0.39 mM and 0.22 mM, respectively. And this enzyme had a strong affinity for L-xylosone and hydroxypyruvaldehyde. The enzyme showed its optimal activity at pH 6.5-7.5 and at $40^{\circ}C$. $^1H$-NMR spectroscopic analysis of enzymic reaction showed that this enzyme catalyzes intramolecular proton transfer.