• 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.

• Applied Biological Chemistry
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• v.50 no.2
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• pp.107-110
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• 2007

The putative laccase gene, yacK of Escherichia coli, K-12 is not expressed in lab culture conditions. The laccase gene was amplified by PCR and subcloned into pET28a vector. The laccase overproduced in E. coli harboring pET28a was purified by His-affinity column chromatography. The purified laccase, which has the apparent molecular weight of 55,000 on the SDS-polyacrylamide gel showed enzyme activity on the guaiacol solution and agar plate. Optimum temperature and pH were around 65$^{\circ}C$ and 5.0, respectively.

• Korean Journal of Microbiology
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• v.21 no.4
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• pp.221-228
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• 1983

An improved procedure for the rapid purification of glucose-6-phosphate dehydrogenase from extracts of Saccharomyces cerevisiae was developed by using affinity chromatography. Among six affinty media tested, $NADP^+ -agarose$ and Affi-gel Blue were more effective than others (i.e., Affi-gel Red, AMP-agarose, ATP-agarose, and $NAD^+ -agarose$). Conditions to desorb the enzyme bound to the affinity media were examined to increase the purity as well as yield. The best result was obtained when the column was developed with a linear gradient of KCl (0-1.0M). In case of Affi-gel Blue, introduction of $NAD^+$ (15mM) washing step prior to the salt gradient was most effective to remove $NAD^+ -binding$ proteins. For a large scale preparation of G-6-P dehydrogenase higher recovery was obtained by Affi-gel Blue than $NADP^+ -agarose$, however, the purity of the enzyme was decreased by 10 times if the former was used as the affinity medium. The capacity of Affi-gel Blue for G-6-P dehydrogenase was found to be 5 times higher than that of $NADP^+ -agarose$. Furthermore Affi-gel Blue could be reused repeatedly and its preparation is relatively easier and less expensive than $NADP^+ -agarose$.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.201-204
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• 2003

Glycopeptide antibiotics, teicoplanin was purified from a mutant strain of Actinoplanes teichomyceticus ATCC31121, A. teichomyceticus MSL2211. We developed a simple procedure to separate and purify the teicoplanin from the fermentation broth. Teicoplanin was purified by two-step purification system, hydrophobic adsorption and sugar affinity chromatography in combination with HPLC analysis based on the properties of hydrophobic acyl chain and sugar moiety in teicoplanin. Teicoplanin was separated from the culture broth by Diaion HP-20 and further purified by concanavalin A affinity column chromatography. As an adsorbent resin, Diaion HP-20 in broth eliminated toxic effects on growth, reduced feedback repression of teicoplanin production, and assisted In rapid recovery of teicoplanin. The teicoplanin displayed the final yield of 80% and 95% of purity.

• YAKHAK HOEJI
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• v.52 no.1
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• pp.79-84
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• 2008

Alismatis Rhizoma has been used as diuretics and antiphlogistics in the Chinese oriental medicine. A trypsin inhibitor was isolated from Alismatis Rhizoma using DEAE ion exchange column, trypsin affinity column, and FPLC chromatography, and its activity and characteristics were studied. The purifed Alismatis Rhizoma trypsin inhibitor (ARTI) was estimated to be about 22 kDa. The sequence determination on N-terminal amino acid residues and 84 amino acid residues has been completed, yet no homology has been found with trypsin inhibitors reported at NCBI. ARTI did not show inhibitory activities on chymotrypsin and elastase, however it exhibited a significant inhibitory activity on bovine trypsin, and formed a complex with rat liver 10-formyltetrahydrofolate dehydrogenase.

• Journal of Food Hygiene and Safety
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• v.8 no.4
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• pp.251-254
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• 1993

Extraction and Clean-up procedures coupled with quantitation by high performance liquid chromatography(HPLC) was evaluated for the detection of 4 aflatoxins, B1, B2, G1 and G2, in peanut butter. The Sep-pak clean-up method showed poorer separation and repeatability than did the modified DeVries' and an immunoaffinity column clean-up methods. No significant difference of detected aflatoxins between the affinity column clean-up and modified DeVries' method. The coefficients of variation for the 4 aflatoxins were ranging from 6.3∼32.3 by the modified DeVries' method and 5.3∼9.8 by the affinity column clean-up.

• Membrane Journal
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• v.8 no.1
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• pp.50-58
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• 1998

Protein affinity membranes were prepared via coating of chitosan gel on the porous flat and hollow-fiber polysulfone membranes, followed by the immobilization of the reactive dye (Cibacron Blue 3GA) to the chitosan gel. Maximum protein binding capacity of these affinity membranes was about 70 $\mu{g/cm}^2$. Using the affinity flat membrane module, the elution chromatography of human serum albumin (HSA) was performed to determine the optimum condition of eluent buffer. The optimum condition of eluent was the universal buffer solution of 0.06 M concentration containing 1 M KCl at pH 10. For the frontal chromatography of HSA using the flat module, the dynamic protein binding capacity was rapidly decreased from the equilibrium values with increasing flow rate and HSA concentration of the loading solution. However, in the case of hollow-fiber module, the dynamic binding capacity was maintained an equilibrium value without depending on the operating conditions. These results showed that the hollow-fiber module was more effective than the flat module as an affinity chromatography column.

• Journal of Dairy Science and Biotechnology
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• v.15 no.1
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• pp.1-9
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• 1997

This study was conducted to efficiently separate the Ig from Korean native cattle colostrum and to utilize them as an immunogen for the production of antibodies aginst rabbit. The results obtained were as follows : 1. About 84% of Ig G could be separated from Korean native cattle colostrum by·gel filtration using Superose 12 column on HPLC. The separation profile of Korean native cattle colostral immunoglobulin was similar that of Holstein colostral Ig. 2. Separation of Korean native cattle colostral Ig by anion exchange chromatography using Mono Q column on HPLC was poor resolution chromatographic pattern. 3. Hi-Trap Protein G column showed better results than the Protein A Sepharose CL-4B column in the Ig G binding capacity from Korean native cattle colostral Ig. 4. Protein G Sepharose Fast Flow system resulted in higher Ig g binding capacity as the industrial size scale-up approach. 5. Sufficient titer reaction of antibody to Korean native cattle colostral Ig G was confirmed by ELISA.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.221-224
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• 2001

Effect of hyperosmotic pressure on growth of recombinant Chinese hamster 。 vary cells and Erythropoietin (EPO) production was investigated. Cells were cultivated in batch modes at various osmolalities. When the osmolality increased from 314 to 463mOsm/Kg, specific EPO productivity (qp) was increased up to 1.6-fold but cell growth was inhibited. EPO has a complex oligosaccharide structure that plays an important role in biological activity in vivo. To investigate the influence of hypoerosmotic pressure on the glycosylation, structural analysis of oligosaccharide was calTied out. Recombinant human EPO was produced by CHO cells grown under various osmotic pressure and purified from culture supernatants by heparin-sepharose affinity column and immunoaffinity column. N-linked oligosaccharides were released enzymatically and isolated by paper chromatography. The isolated oligosaccharides were labeled with fluorescent dye, 2-aminobenzamide and analyzed with MonoQ anion exchange chromatography and GlycosepN amide chromatography for the assignment of GU (glucose unit) value. Glycan analysis by HPLC showed that neutral (asialo) oligosaccharide was increased slightly with an increase in osmolality. In portion of sialylated glycan, total relative amount of mono- and di-sialyated glycan was increased but that of tri- and tetra-sialylated glycan decreased as osmolality was increased.

• Parasites, Hosts and Diseases
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• v.29 no.1
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• pp.1-8
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• 1991

Out of many component proteins in crude saline extract of Spirometra mansoni plerocercoid (sparganum) , 36 kDa and 29 kDa proteins were found to be the most antigenic and were already purified by immunoaffinity chromatography using monoclonal antibody as a ligand. In this study, a single step purification of these potent antigenic proteins of sparganum extract was investigated. When the crude saline extract was charged to gelatin-Sepharose 4B affinity column, 36 kDa and 29 kDa protein fractions were bound. SDS-polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE/immunoblot confirmed that the bound protein to gelatin was serologically pure. When evaluated by ELISA with patients sera, the purified protein of 36 and 29 kDa also showed improved antigenicity.