• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.34-38
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• 2003

For the production of $B^{30}-homoserine$ insulin analog as a novel anti-diabetic drug, the fermentative study was attempted for the maximal gene expression of HTS-fused $B^{30}-homoserine$ insulin precursor in the recombinant Escherichia coli cells. In a batch fermentation, the maximal production of insulin precursor as much as 38.95 mg/L-h, which occupied more than 12.8% of total cell protein. was achieved when the gene expression was induced by 0.5 mM IPTG at the middle logarithmic growth phase. The HTS-fused $B^{30}-homoserine$ insulin precursor was recovered from a batch culture through the processes of cell harvest, collection of insoluble fraction after sonication and purification by nickel affinity column chromatography. The isolated insulin precursor was 14 mg/L with a recovery yield of 35.9% of expressed gene product. The insulin A and B chain mixture was recovered after the insulin precursor was subjected to CNBr cleavage and purified by nickel affinity column chromatography. The isolated insulin chains were then sulfitolyzed with sodium thiosulfat and sodium tetrathionate, and reconstituted to insulin analog with ${\beta}-mercaptoethanol$, followed by purification with CM-Sepharose C-25 column chromatography.

• Biomedical Science Letters
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• v.4 no.1
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• pp.35-42
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• 1998

The effects of CRP purified from human ascites fluid on phagocytic activity of the human macrophage were investigated. CRP was purified using affinity chromatography including absorption on p-diazonium phosphocholine or C-polysaccharide coupled sepharose 4B and gel filtration on hydroxylapatite column chromatography. Macrophage was separated ficoll hypaque gradient density and absorption method, and then was confirmed phagocytic uptake test using latex method. CRP was able either to inhibit or to enhance phagocytic activity of human macrophage against bacteria in vitro. The effects of CRP on phagocytic activity of human macrophage were in time and dose-dependent manners. The additional sequence of reaction mixture against bacteria in vitro shows a threshold stimulus on the activation of phagocytic response upon the CRP.

• 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.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.344-349
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• 2004

A gene encoding inulin-degrading enzyme of Bacillus sp. snu-7 with ORF of 1536 nucleotides was cloned. And it was overexpressed as His-tagged protein in E. coli BL21(DE3) pLysS using pRSET B vector containing mature enzyme sequence. Maximum enzyme production was achieved by IPTG (0.1 mM) induction at $OD_{600}$ 1.2 and $30^{\circ}C$ followed by 6 h incubation. The expressed protein purified through immobilized metal affinity chromatography showed molecular mass of 60 kDa on SDS-PAGE. Results of thin-layer chromatography using inulin as a substrate showed the enzyme to be an exotype inulinase capable of producing only monomeric fructose as a product. $K_m$ and $k_{cat}$, for the hydrolyses of inulin and sucrose were $2.28\pm0.08$ mM and 358.05$\pm$20.38 $min^{-l}$, and 22.02$\pm$0.41 mM and 4619.11$\pm$215.12 $$min^{-1}, respectively. Optimal activity of the exoinulinase occurred at pH 7.0 and $50^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.27 no.5
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• pp.983-989
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• 2017

NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of $NADP^+$ in the affinity chromatography process. In the present study, the rat NPR clone containing a $6{\times}$ Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using $Ni^{2+}$-affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochrome c and nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.

• Journal of the Korean Chemical Society
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• v.23 no.3
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• pp.165-174
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• 1979

A purification technique of glucose oxidase was developed. Using the gluconyl-${\omega}$-aminohexyl Sepharose affinity chromatography, it was partially purified 14.6 folds with 79.7% yield. With the combination of the affinity chromatography and Sepharose 6B gel filtration, the enzyme was purified 27.2 folds from the broth with 74.1% yield. The final purified preparation showed 90.83 U of glucose oxidase activity per mg of protein and a single band by 7% polyacrylamide gel electrophoresis. The absorption spectrum and substrate specificity of the enzyme were studied and the fianal preparation showed the optimal pH between 5.6 and 6.0, the optimal temperature at $40^{\circ}C$, $8.5{\times}10^{-3}M$ of $K_m$ for D-glucose, and 3.43 kcal/mole of the activation energy.

• Korean Journal of Microbiology
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• v.38 no.4
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• pp.241-246
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• 2002

A gene coding for endo-$\beta$-1,3-1,4-glucanase of Bacillus circulans was subcloned into Escherichia coli Ml5 using pQE30 as an expression vector. Endo-$\beta$-1,3-1,4-glucanase produced by the recombinant expression plas-mid pLQ43 was intactly purified to a single protein through a nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography method. The molecular mass of the purified enzyme was estimated to be 28 kDa by SDS-PAGE. The optimum pH and temperature of the enzyme activity were pH 6.8 and $60^{\circ}C$, respectively. This enzyme was fairly stable in the pH ranging 5.5~7.5 and at the temperatures lower than $55^{\circ}C$. The enzyme appeared to be sensitive to most of the metal ions, especially to $Hg^{2＋$, and also to methanol, ethanol, isopropanol or 1-butanol at a concentration of 10%(v/v).

• The Korean Journal of Zoology
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• v.32 no.4
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• pp.420-428
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• 1989

Pseudogobio esocinus의 심장, 신장 및 간 조직은 하부단위체 C를 함유하는 젖산수소이탈효소를 갖고 있음이 확인되었다. 하부단위체 A 및 B에 대한 유전자들의 조직 발현은 다른 포유동물의 것과 유사하였으며 분자량은 140,000 정도로 추정되었다. Oxamate gel을 사용한 chromatography결과 A4 동위효소는 NAD+보다는 column buffer에 의해 용출되었다. B4 동위효소는 CM-Sepharose column을 사용하여 부붙 정제되었다. B4 동위효소는 물론 A4 동위효소도 고농도의 Pyruvate에 의해 저해되었다. A4 동위효소의 affinity chromatography 상 행동과 Pyruvate 저해 정도로 보아 A4 등위효소는 B4 동위효소 두 역학적으로 유사하다고 사료된다. P. esainus A4 동위효소에 대한 항체는 mouse A4 등위효소와 반응하지만 동종의 B4 동위 효소와는 반응하지 않는 특성으로 보아 하부단위체 B는 진화과정에서 보존성이 낮은 것으로 사료된다. Three tissues of heart, kidney and liver of a primitive cvprinid Pseudogobio esocinus were found to have lactate dehydrogenase isozyme(5) containing subunit C. Tissue expressions of genes for subunits A and B were similar to those of mammalian species. Molecular weight of the isozymes were estimated to be 140,000 approximately. Affinity chromatography of the isozymes on the immobilized oxamate gel revealed that A4 isozyme was not elected in NAD+ but in column buffer. B4 isozune was isozpnatically purified by subjecting kidney extract to a CM-Sepharose column. Ae isozvme as well as B4 isozvme was inhibited by high concentrations of pyruvate. The affinity chromatographic behavior and susceptibility to pyruvate inhibition of the A4 isorpne suggest that A4 isozwne is similar to B4 isozyme kinetically. Antibodies against p. esocinus A4 isogyme reacted with mouse At isozyme but not with p. esocinus B4 isogyme, reflecting that subunit B is less conservative in its evolution.

• 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.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.33 no.1
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• pp.55-59
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• 2000

A specific and sensitive sandwich enzyme-immunoassay (EIA) using Avidin-Biotin complex was developed for the measurement of GTH II levels in pituitary content and pituitary cell culture medium of the rainbow trout-(Oncorhpchus mykiss). Biotin-salmon GTH II rabbit IgG (sefondary antibody) wai purified by a protein A sepharose affinity chromatography column and that was biotinylated by using Biotin-N-hydroxysuccinimide ofter (BNHS). Non-biotin salmon GTH II rabbit IgG (first antibody) was obtained only through a protein A sepharose affinity chromatography column. The assay was performed by the so-called 'sandwich' method using a microtiter plate, A dose-response curve was obtained between $0.12 to 125 ng/ml$ of salmon GTH II. The displacement curves for pituitary extraction and pituitary cell culture medium of testosterone-treated rainbow trout were Parallel to the standard curie. The intra-assay and inter-assay coefficients of variation (CV) were $8.2{\%} (N=5) and 12.5{\%} (N=6)$, respectively, This assay system was used to measure the amount of GTH II that accumulated in the culture medium of dispersed pituitary cells in testosterone-treated immature rainbow trout, The accumulation was increased with the amount or salmon gonadotropin-releasing hormone. GTH II values determined by the present method were well correlated with those determined by radioimmunoassay. As a result, this assay system was found to be suitable for the measurement of GTH II for pituitary extraction and pituitary culture medium in many salmonid fishes.