• YAKHAK HOEJI
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• v.30 no.5
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• pp.220-227
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• 1986

Polyphenol oxidase was purified from an extract of tea leaf by ammonium sulfate fractionation followed by Sephadex G-150 column chromatography, which resulted in a 67-fold increase in specific activity. The enzyme had optimum pH 6.5 and was relatively heat stable. The substrate specificity of the tea leaf PPO showed high affinity toward pyrogallol and catechol. Potassium cyanide, sodium diethyldithiocarbamate, L-cysteine, 2-mercaptoethanol and ascorbic acid were potent inhibitors.

• Proceedings of the Membrane Society of Korea Conference
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• 1998.04a
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• pp.120-123
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• 1998

1. 서론 : 생물기술이 급속히 발전함에 따라 다양한 생물제품들(예, 단백질, 효소, 항생제 등)이 생산되고 있으나, 그 생산량은 극단적으로는 기질용액 1g당 10$^{-8}$g 정도로까지 낮아, 이를 고순도로 정제하기 위해서는 다단계의 공정이 필요하여 분리정제의 비용이 크다. 따라서 생물제품의 산업적 생산이 경제성을 갖기 위해서는 생물제품을 보다 경제적으로 분리정제할 수 있는 효율적인 공정개발이 필수적이다. (생략)

• Microbiology and Biotechnology Letters
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• v.16 no.5
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• pp.335-342
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• 1988

An anion exchange chromatography was employed for the purification of mouse monoclonal antibodies from ascitic fluid and in vitro cultivation media. After cultivation of hybridomas, Alps 25-3, HCGK, A4W, and KW, producing IgG1, the culture supernatants were harvested by centrifugation, precipitated with 50-60％ ammonium sulfate, and dialyzed against 0.025 M Tris-HCI buffer (pH 8.2). Then the dialyzed samples were loaded into a DEAE-Trisacryl M anion exchange column. Monoclonal antibodies bound to the DEAE-Trisacryl M were eluted with 0.025 M Tris-HCI buffer (pH 8.2) containing 30-40 mM NaCl. In ammonium sulfate precipitation, the recovery of the monoclonal antibody was shown to be 90％ and 84％ from in vitro culture media containing 10％ and 2％ fetal bovine serum, respectively. On the other hand, the pretreatment by ultrafiltration enhanced the yield up to 91％ whereas the purity was lower than that by ammonium sulfate treatment. Subsequently, in the DEAE-Trisacryl M chromatographic separation, the purities and recoveries of all the monoclonal antibodies from both the in vitro culture supernatants and ascitic fluids were 70-80％ and 65％ respectively. The monoclonal antibody, Alps 25-3 could be further purified with a purity of 95％ through an immunoadsorbent chromatography.

• Journal of Plant Biology
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• v.35 no.2
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• pp.99-106
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• 1992

The endosperm protein, vicilin, of ginseng (Panax ginseng C.A. Meyer) was purified by ammonium sulfate precipitaion, gel permeation and ion exchange column chromatography. Vicilin is a glycoprotein composed of 2 subunits with molecular masses of 55,000 (large subunit) and 44,000 (small subunit). The anti-vicilin antibody was raised in rabbit, and purified by DEAE Affi-Gel Blue affinity chromatography. The endosperm cells of the seed were reacted with this anti-vicilin antibody and colloidal gold conjugated secondary antibody. Gold particles were labelled on the elaborating granules of Golgi complex, electron-dense granules and protein bodies in the endosperm cells. These results indicated that the vicilin, which was synthesized in rough endoplasmic reticulum and transported to Golgi, was elaborated in saccules of the Golgi and then transported into protein bodies by electron-dense granules.anules.

• BMB Reports
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• v.37 no.5
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• pp.556-564
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• 2004

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2% glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.

• Horticultural Science & Technology
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• v.29 no.3
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• pp.255-259
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• 2011

Polyphenol oxidases (PPO) are copper-containing enzymes responsible for tissue browning in fruits and vegetables including potato, apple and pears. Although these enzymes have been studied for many years, their physiological roles in plants are not yet clear. Therefore, these enzymes need to be purified to characterize further from potato tubers. The classical methods used for the purification of PPO involve several steps. So in this study, we developed a one-step chromatography process for the potato tuber PPO purification. After removal of salts from dissolved ammonium sulfate precipitates of potato tuber extracts using Sephadex-G50 gel filtration, affinity chromatography was carried out on NHS-activated Sepharose 4B using p-aminobenzoic acid as a ligand. The purified enzyme was confirmed by silver staining and a zymogram. The optimum temperature and pH for the purified potato tuber PPO were $15^{\circ}C$ and pH 6.0, respectively. The results obtained in the present study will aid to evaluate PPO from various fruits and vegetables.

• The Korean Journal of Zoology
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• v.29 no.4
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• pp.283-293
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• 1986

For the preliminary step to make and characterize the monoclonal antibodies of human alpha-fetoprotein (AFP) was purified from 534g of human fetal tissues through the procedures of tissue extraction, DEAE-cellulose, concanavalin A-Sepharose, Cibacron Blue F3GA-agarose and immunoadsorbent affinity chromatography. The isolated AFP preparation showed a single band on polyacrylamide gel electrophoresis and a single precipitin are against rabbit anti-human cord serum and anti-human AFP on immunoelectrophoresis. Our AFP also displayed a single band on SDS-polyacrylamide gel electrophoresis. The recovery of AFP was 8.76mg total.

• Journal of the Korean Chemical Society
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• v.40 no.1
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• pp.72-80
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• 1996

A chitinase-producing bacterium, Serratia marcescens JM, was isolated from a seashore muds. A chitinase was purified by ammonium sulfate precipitation, affinity adsorption, hydroxylapatite and sephadex G-200 column chromatography. The chitinase obtained from Serratia marcescens JM was purified 42.2 folds with the overall yield of 7.1%. The purified chitinase showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 59,000 and the apparent kinetic parameters $K_m\;and\;V_{max}$ for the purified chitinase were 5.17 mg/mL and 39.8 unit/mL, respectively. The optimum pH and temperature of the purified chitinase were 7.0 and 50$^{\circ}C$, respectively and the purified enzyme was stable on pH 7.0 up to 50$^{\circ}C$. The enzyme were activated by $Cu^{2+},\;Ca^{2+}\;and\;Mg^{2+}$ and inhibited by $Hg^{2+}$ respectively. In addition, Cysteine increased the chitinase activity and EDTA, MIA, PCMB and SDS inhibited enzyme activities. Major cations, $MG^{2+},\;Ca^{2+},\;K^+\;and\; Na^+$ present in seawater slightly stimulated the chitinase activity.

• YAKHAK HOEJI
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• v.47 no.6
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• pp.410-416
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• 2003

Autotaxin(ATX) was originally purified from conditioned media of A2058 human melanoma cells and shown to be a potent cell motility-stimulating factor, possessing a type II nucleotide pyrophosphatase/phosphodiesterase (NPP2) activity. Recombinant ATX has recently demonstrated that human plasma lysophosholipase D is identical to ATX and uses lysophosphatidylcholine as a substrate to mediate various biological functions including tumor cell growth and motility through G-protein coupled receptor. However, despite pivotal roles of ATX on physiological or pathophysiological states, the production of ATX is solely depends on complicated purification method which employs multiple column steps, but resulted in very poor yield. This limited the use of ATX for extensive analysis. We, therefore, expressed six histidine-tagged recombinant human ATX(His-ATX) in High Five TM insect cells to improve the generation of ATX and to make simple the purification of ATX. The signal sequence of the human ATX gene was truncated and replaced with sequence of insect cell secretion signal within expression vector. In addition, codons for six histidines were added to the C-termini of 120kDa ATX cDNA construct. A simple purification scheme utilizing two-step affinity column chromatography was designed to purify His-ATX to homogeneity from the culture supernatant of transfected insect cells. Homogenous His-ATX was detected and isolated from the concentrated insect cell medium using concanavalin A agarose and nickel affinity chromatography. Purified His-ATX was in full length with ATX capacity. A combination of this expression system and purification scheme would be useful for production and purification of high-quality functional ATX for research and practical application of multiple functional motogen, ATX/NPP-2.

• Applied Biological Chemistry
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• v.41 no.7
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• pp.505-509
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• 1998

Chitinase, which is one of the pathogenesis-related proteins, was examined from Rehmannia glutinosa. Rehmannia chitinases were classified into basic and acidic groups by chitin affinity column chromatography. According to the chitinase pattern by native PAGE, basic chitinases of low Rf values were extracted more under an acidic condition (pH 2.9) than a basic condition (pH 8.8), while acidic chitinases having high Rf values were mainly detected in basic extracts. There were in total seven chitinase isoforms of three basic and four acidic isoforms in Rehmannia. The range of molecular weight of Rehmannia chitinase was from 28 to 32 kD. It showed the possibility of tissue specific expression of acidic chitinases and basic chitinases were mainly detected in root.