• 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.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.196-203
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• 2002

A novel process for urokinase purification was studied using p-aminobenzamidine as the ligand and sepharose 4B as the matrix. The adsorption, washing, and elution conditions were optimized by an unusual method. An adsorption buffer containing 2.5 M NaCl and $1\%$ Tween 80 facilitated the adsorption of urokinase on the affinity media and prevented contaminants from binding to the p-aminobenzamidine affinity gel. It was found that $5\%$ Tween 80 removed most of the contaminants from the affinity column. A 0.2 M glycine elution buffer containing 0.5 M NaCl (pH 3.0) was found to have a strong elution ability with a high recovery and purity of urokinase. A crude urokinase material of231 IU/mg protein from human urine was purified to 124,300 IU/mg protein with a purification factor of 538 and yield of $86.7\%$. As a result, a high purity urokinase was obtained with only a single affinity chromatography step. The purification process was successfully scaled-up to a 2-1 chromatography column. The resulting urokinase eluate could be directly lyophilized, thereby complying with Chinese pharmacopoeia (1995 version) standards.

• Biomedical Science Letters
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• v.2 no.1
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• pp.41-48
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• 1996

The C-reactive protein(CRP) from ascitic and pleural fluid was purified using calcium dependent affinity chromatography of CNBr activated Sepharose-4B covalently coupled to p-diazonium phenylphosphorylcholine(DPPC) and hydroxylapitite chromatography. Polyclonal antibody was prepared from rabbit by immunizing the purified CRP. Specific immunoglobulin G was isolated using affinity chromatography and coupled to microparticles. A sensitive microparticle-based immunoassay was developed to measure CRP within 3 mins. The detection range was between 0.5mg/dl) and 20mg/dl in serum, showing strong response in the range of 0.7~2.9 mg/dl, weak response in 5.0~13.2 mg/dl and zone phenomenon over 28mg/dl. The average value of CRP in 74 samples was 3.8mg/dl and most of the values were lower than 10mg/dl .The CRP values of serum samples were determined by our microparticle-based immunoassay, and were compared with those obtained using the other commercial products(B Co., France and I Co., Japan). Good correlations were shown between the values obtained by our developed microparticle-based immunoassay system and those by other commercial products. All performance characteristics evaluated make our developed microparticles-based immunoassay suitable for a simple, rapid, and reliable screening of CRP in serum.

• Proceedings of the Membrane Society of Korea Conference
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• 2000.11a
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• pp.115-118
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• 2000

• Bulletin of the Korean Chemical Society
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• v.13 no.2
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• pp.208-210
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• 1992

• Journal of Microbiology and Biotechnology
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• v.19 no.10
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• pp.1191-1196
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• 2009

The production of Streptomyces griseus trypsin (SGT) by S. griseus IFO13350 transformed with the expression vector pWHM3-TR1R2, containing sprT encoding SGT and the two positive regulatory genes sgtR1 and sgtR2, was investigated in various media. Cultivation in Ferm-0 gave 1.4 times more trypsin activity than in C5/L medium. In addition, replacement of 2% glucose and 1% skim milk in Ferm-0 with 2% dextrin and 1% tryptone (designated Ferm-II) enhanced trypsin activity 4.1-fold. To simplify the purification process, the supernatant from the S. griseus transformant cultured in Ferm-II medium was fractionated with ammonium sulfate (25-55%), then subjected to Hitrap Benzamidine FF affinity column chromatography. The specific activity of SGT purified by one-step chromatography was 69,550 unit/mg protein and the overall purification yield was above 8%, indicating that this method is more effective than those previously reported. Purified SGT was most active at pH 8.0 and $50^{\circ}C$, and it maintained activity between pH 7.0 and 9.0 and at temperatures up to $70^{\circ}C$. These enzymatic properties are very similar to those of authentic eukaryotic trypsin purified from bovine pancreas.

• Korean Journal of Food Science and Technology
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• v.30 no.1
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• pp.218-223
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• 1998

Casein was hydrolyzed by alcalase to produce iron binding peptide (IBP). IBP was effectively separated from casein hydrolysates by immobilized $Fe^{3+}$ affinity chromatography and further purified by reverse phase chromatography. $25,\;50\;and\;100\;{\mu}g/mL$ of IBP solubilized $4.2,\;5.7\;and\;7.1\;{\mu}g$ of ferric at duodenum condition $(pH\;6,\;37^{\circ}C)$, respectively. According to the result of MALDI analysis, molecular weight of IBP was determined to 2,175 dalton. IBP was mainly composed of proline (24.5 mol%), lysine (15.7 mol%), and glutamine or glutamic acid (14.9 mol%) and its N-terminal sequence was Met-Ala-Pro-Lys-His. According to the information obtained from molecular weight, amino acids composition and N-terminal sequence of IBP, it was evident that IBP was from f102-119 of ${\beta}-casein$.

• Bulletin of the Korean Chemical Society
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• v.18 no.6
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• pp.648-653
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• 1997

Acetolactate synthase was purified from Escherichia coli MF2000/pTATX containing Arabidopsis thaliana acetolactate synthase gene. Purification steps included DEAE cellulose ion exchange column chromatography, phenyl sepharose hydrophobic column chromatography, hydroxylapatite affinity column chromatography, and Mono-Q HPLC. Molecular weight was estimated to be ∼65 KDa and purification fold was 109 times. The enzyme showed a pH optimum of 7 and the $K_M$ value was 5.9 mM. The purified enzyme was not inhibited by any of the end products, valine, leucine, and isoleucine.

• Applied Biological Chemistry
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• v.49 no.3
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• pp.180-185
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• 2006

${\alpha}$-Galactosidase was partially purified from the culture filtrate of Debaryomyces sp. by Mannobiose-Sepharose affinity column chromatography. The galactosidase exhibited maximum activity at pH 4.0 and $60^{\circ}C$, and was stable in the pH and temperature ranges of 3 to 4.5 and 30 to $50^{\circ}C$, respectively. The enzyme was inhibited by $Hg^{2+}\;and\;Ag^{2+}$. The enzyme activity was not affected considerably by treatment with other metal compounds. The enzyme hydrolyzed melibiose to galactose and glucose, raffinose to galactose and sucrose, and $Gal^3Man_3$ ($6^3-{\alpha}$-galactosyl-1,4-mannotriose) to galactose and mannotriose. On the contrary, it could not hydrolyze $Gal^3Man_4$ ($6^3-{\alpha}$-galactosyl-1,4-mannotetraose).

• Journal of the Korean Society of Food Science and Nutrition
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• v.10 no.1
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• pp.67-76
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• 1981

By using the paper chromatograph technique affinity of amino acids to the mercury was compared with that of sodium thiosulfate, 2,3-dimercaptopropanol(BAL) and inosinic acid. Among the amino acids of testment three acids (Cysteine, Cystine and Methionine) which posses sulfide or disulfide radical exhibited a spot which combined more apparently with the mercury than other amino acids. However, in the mixed solutions of thiosulfate, BAL or inosinic acid with those amino acid and mercury, the mercury was moved into thiosulfate, BAL or inosinic acid spot. It was clear from the results that affinity of sodiume thiosulfate, BAL and inosinic acid to the mercury is higher than that of amino acids.