• KSBB Journal
• /
• 제9권2호
• /
• pp.115-121
• /
• 1994

240ml의 시료를 정제할 수 있는 30channel의 preparative-scale 자유유동전기이동 분리장치를 제작한 후 렌즈콩으로부터 lentil lectin(LcH)을 전기 집속법으로 분리하였다. 분리된 각 분획을 PAGIEF젤에서 silver staining했을 때 불순물이 전혀 검출되지 않을 정도의 고순도 lectin을 얻을 수 있었다. 이 때 ampholyte로서 HEPES(50mM) -Tris(50mM) -urea(3M) 등을 사용하였으며 50mM Histidine(pI 7.65)인 경우 가장 분리도가 가장 좋았다. LcH는 보통의 조건하에서 LcH-A와 LcH-B의 두가지 순수한 형태로만 나타났으며 따라서 이 장치를 사용하여 다단 분리 정제를 할경우 두 성분의 완전한 분리도 가능함을 보여 주었다.

• 한국미생물·생명공학회지
• /
• 제23권4호
• /
• pp.411-415
• /
• 1995

The dextranase (EC 3.2.1.11) produced by Aspergillus ustus GR-98 was purified by the following sequential methods; salting-out and dialysis, gel filtration on BIO-GEL P-100, ion exchange chromatography on DEAH-cellulose, affinity chromatography on hydroxyapatite, and preparative electrophoresis. Three active fractions, dextranases 1, 11 and 111, were isolated in electrophoretically pure states, and specific activities of the dextranases were 1,276, 1,154 and 1,125 units/mg, the degrees of yield were 9.0, 3.6 and 2.2%, having 145, 131.1 and 127.8 times as those of culture filtrate in degree of purification, respectively. The enzyme purity was confirmed by the PAGE, SDS-PAGE and get permeation-HPLC.

• 한국동물학회지
• /
• 제37권1호
• /
• pp.66-75
• /
• 1994

Juvenile hormone binding protein was identified in the hemolymph of fifth instar larvae and purified using column chromatography. Hemolymph was mixed with [3H] JH-III and electrophoresed on 691 NON-SDS gel, indicating that radioactivity peak appears at Rf value of 0.55. Gel filtration showed two radioactivity peaks equivalent to bound and free [3H]JH-III, respectively. JHBP was purified from hemolymph through gel filtration (Sephadex G-100), anion exchange chromatosraphv (DEAE Sepharose CL-6B), chromatofocusing chromatographv (PBE 94) and preparative electrophoresis.

• 한국동물학회지
• /
• 제19권2호
• /
• pp.63-70
• /
• 1976

免疫抗體分子의 基本 構造成分인 H-chain, L-chain외에 重合抗體分子 속에서만 발견되는 이른바, J-chain의 構造와 機能을 밝히기 위한 前段階로 J-chain의 純粹分離를 試圖하였다. 우선 多發性骨髓腫 患者의 血淸으로부터 重合型 IgA를 純粹分離한후, 이를 환원시켜 L-J-chain 混合物을 얻은 다음 3가지 方法, 1) 제조용 디스크 전기영동법 2) 脫鹽 透析法, 3) 이온-교환 크로마토그라피법으로 J-chain을 순수분리할 수 있었다. 위 3가지 방법으로 분리한 J-chain의 物理化學的 및 生化學的 성상은 동일하였으며, 사용한 세가지 방법 중 脫鹽 透析法이 가장 간편하고 효과적인 方法임을 알았다.

• 한국식품영양과학회지
• /
• 제12권4호
• /
• pp.387-394
• /
• 1983

zymolyase(${\beta}-1$, 3-glucanase)의 효모(酵母) 세포벽(細胞壁)에 대한 용해작용(溶解作用)을 촉진(促進)시키는 인자(因子)를 zymolyase의 조효소(粗酵素)로부터 분리(分離)하여 Sephadex G-75 gel 여과(濾過) 및 조제용(調製用) PAG-전기영동법(電氣泳動法)에 의하여 전기영동적(電氣泳動的)으로 균일(均一)하게 정제(精製)하였다. SDS-PAG 전기영동법(電氣泳動法)에 의하여 측정한 정제(精製) 인자(因子)의 분자량(分子量)은 40,500이었고, 등전점(等電點)은 pH 9.6이었다. 정제(精製)된 촉진인자(促進因子)는 395개(個)의 아미노산 잔기(殘基)로 구성(構成)되는 단일(單一) polypeptide의 염기성(鹽基性) protease임이 밝혀졌으며, 이 protease의 흡광계수(吸光係數)($E_{208,cm}^{1%}$)는 11.9, 분자흡광계수(分子吸光係數)(${\varepsilon}_{280,}mole/l$)는 $4.83{\times}10^4$이었다.

• Animal cells and systems
• /
• 제3권2호
• /
• pp.173-180
• /
• 1999

A blue biliprotein, insecticyanin (INS), has been purified from the last instar larval hemolymph of Agrius convolvuli by ultracentrifugation, Sephadex G-100 gel permeation chromatography, and preparative electrophoresis. The molecular mass of INS was estimated to be 26 kDa and the N-terminal sequence of INS revealed high similarity to that of Manduca sexta. Results of Western blotting and autoradiography indicated that INS is synthesized by the epidermis and released into the hemolymph. In contrast to the INS reported in other insects, Agrius convolvuli INS contained a small amount of lipid, predominately consisting of triacylglycerol. Subcellular localization of INS was determined using protein-A gold particles linked to secondary antibodies (anti-rabbit Ig). INS was heavily accumulated in the cytoplasmic inclusion body (CIB). CIBs showed a variety of shapes from rod to globule and generally surrounded the nucleus. They were mostly located near the basement membrane and especially abundant in the intersegmental membrane.

• 한국미생물·생명공학회지
• /
• 제24권3호
• /
• pp.282-289
• /
• 1996

2,3-DHBP dioxygenase was purified from E. coli CK1092 carrying the pcbC gene, which was cloned from 4-chlorobiphenyl-degrading Pseudomonas sp. P20. Purification of this enzyme was done by acetone precipitation, DEAE- Sephadex A-25 ion exchange chromatography, and preparative gel electrophoresis. The molecular weight of subunit was 34 kDa determined by SDS-PAGE, and that of native enzyme was about 270 kDa. It suggests that this enzyme consist of eight identical subunits. This enzyme was specifically active against only 2,3-DHBP as a substrate with 18 $\mu$M of Km value, but not catechol, 3-methylcatechol, 4-methylcatechol and 4-chlorocatechol. The optimal pH and temperature of 2,3-DHBP dioxygenase were pH 8.0 and 40-60$\circ$C. The enzyme was inhibited by Cu$^{2+}$, Fe$^{2+}$ and Fe$^{3+}$ ions, and was inactivated by H$_{2}$0$_{2}$2 and EDTA. The lower concentrations of some organic solvents such as acetone and ethanol don't stabilize the activity of 2,3-DHBP dioxygenase. The enzyme was completely inactivated by adding the reagents such as N-bromosuccinimide, iodine and p- diazobenzene sulfonic acid.

• ALGAE
• /
• 제28권1호
• /
• pp.121-129
• /
• 2013

Intact phycobilisomes from a wild-type red Chondrus crispus and its vegetatively derived green mutant were isolated by centrifugation through a discontinuous sucrose density gradient. Pigment composition was subsequently characterized by spectrophotometry. Vegetative thalli of the two strains grown together for six months in the laboratory resulted in different pigment profiles. Two pigmented phycobilisome bands appeared in the sucrose gradient of the wild-type alga, a purple coloured one, and a pink one, whereas only a single blue band appeared in the gradient of the green mutant. Spectrophotometric and fluorescence analyses identified the phycobiliprotein composition of the purple band as the typical phycoerythrin-phycocyanin-allophycocyanin complement in the wild-type, but there was no detectable phycoerythrin present in the blue band of the green mutant. Sodium dodecyl sulphate, preparative polyacrylamide gel electrophoresis analysis confirmed the presence of allophycocyanin subunits in all extracts, but firm evidence of an R-phycoerythrin linker polypeptide in the blue band was missing. These results highlight the ability of C. crispus to adapt to a phycoerythrin deficiency by adjusting light harvesting pigment ratios.

• Journal of Microbiology and Biotechnology
• /
• 제8권3호
• /
• pp.270-276
• /
• 1998

Extracellular ${\alpha}$-glucosidase was purified to homogeneity from moderately thermophilic Bacillus sp. DG0303. The thermostable ${\alpha}$-glucosidase was purified by ammonium sulfate fractionation, ion-exchange chromatography, preparative polyacrylamide gel electrophoresis (PAGE), and electroelution. The molecular weight of the enzyme was estimated to be 60 kDa by SDS-PAGE. The optimum temperature for the action of the enzyme was at $60^{\circ}C$. It had a half-life of 35 min at $60^{\circ}C$. The enzyme was stable at the pH range of 4.5~7.0 and had an optimum pH at 5.0. The enzyme preparation did not require any metal ion for activity. The thermostable ${\alpha}$-glucosidase hydrolyzed the ${\alpha}$-1,6-linkages in isomaltose, isomaltotriose, and panose, and had little or no activity with maltooligosaccharides and other polysaccharides. The $K_m$ (mM) for p-nitrophenyl-${\alpha}$-D-glucopyranoside (pNPG), panose, isomaltose, and isomaltotriose were 4.6, 4.7, 40.8, and 3.7 and the $V_{max}$(${\mu}mol{\cdot}min^-1$$mg^-1$) for those substrates were 5629, 1669, 3410, and 1827, respectively. The N-terminal amino acid sequence of the enzyme was MERVWWKKAV. Based on its substrate specificity and catalytic properties, the enzyme has been assigned to be an oligo-1,6-glucosidase.

• Journal of Plant Biology
• /
• 제32권1호
• /
• pp.51-65
• /
• 1989

Glycinin is the major storage protein in soybean. It has been known that a molecule of glycinin is composed of 6 subunits, each of which consists of two different kinds of polypeptides, acidic (A) and basic (B) one (NW 39K and 19K, respectively). To study the molecular origin and the relationship of glycinin subunit polypeptides, antibodies against A-and B-polypeptide were obtained by immunizing rabbits with either of the antigens purified by gel filtration and preparative electrophoresis. Each antibody was not only specific for its own antigen polypeptide in soybeans but also recoginzed the precursor which was synthesized in vivo and in vitro. The polyadenylated mRNAs were isolated from immature seeds and leaves and were translated in vitro using wheat germ extract. One of the seed-specific translation products. MW 60K, was identified to be the precursor of glycinin subunit by immunoprecipitation with antibodies against glycinin A- and B-polypeptide. Mature A- and B-polypeptides were not detected in the translte in vitro. These results suggest that the precursor polypeptide is synthesized from the mRNA and is cleaved to yield A- and B-polypeptides which from a glycinin subunit in the cell. Glycinin genes were expressed with the maturation of soybean seeds in a tissue-specific and developmental stage-specific manner.