• 약학회지
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• 제41권5호
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• pp.638-646
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• 1997

The steroid 9${alpha}$-hydroxylase activity has been detected in cytosol fraction, $100,00{\times}g$ supernatant of cell free extract of Mycobacterium fortuitum. The activity was not linear with protein concentration in the assay suggesting 9${alpha}$-hydroxylase is a multicomponent enzyme. The 9${alpha}$-hydroxylase system was partially purified through fractional saturation of ammonium sulfate, strong anion exchange (Mono Q) column chromatography, gel filtration (Superose 12) column chromatography, and testosterone affinity gel chromatography. Ammonium sulfate 50~60% saturated fraction of the cytosol gave 9${alpha}$-hydroxylase activity. For further purification, the half-saturated ammonium sulfate fraction was applied to Mono Q, Superose 12, or affinity gel column. The purification factors of 9${alpha}$-hydroxylase containing fraction after Mono Q, Superose 12, and affinity gel chromatography was 13, 11, and 17 respectively.

• Mass Spectrometry Letters
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• 제7권3호
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• pp.74-78
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• 2016

Targeted glycoproteomics is an effective way to discover disease-associated glycoproteins in proteomics and serial affinity chromatography (SAC) using lectin and glycan-targeting antibodies shows glycan diversity on the captured glycoproteins. This study suggests a way to determine glycan heterogeneity and structural analysis on the post-translationally modified proteins through serial affinity column set (SACS) using four Lycopersicon esculentum lectin (LEL) columns. The great advantage of this method is that it differentiates between glycoproteins on the basis of their binding affinity. Through this study, some proteins were identified to have glycoforms with different affinity on a single glycoprotein. It will be particularly useful in determining biomarkers in which the disease-specific feature is a unique glycan, or a group of glycans.

• 대한화학회지
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• 제53권3호
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• pp.279-285
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• 2009

This paper has described about preparation of $Zr^{4+}$ affinity column based on the poly(styrene-co- gly-cidyl methacrylate) prepared by emulsion polymerization of styrene and glycidyl methacrylate in order to isolate phosphopeptide. The $Zr^{4+}$ ions were introduced after the phophonation of an epoxy group on polymeric microspheres. The successful preparation of $Zr^{4+}$-immobilized polymeric microsphere stationary phase was confirmed through Fourier transform infrared spectra, optical microscopy, scanning electron microscopy, X-ray photoelectron spectra and inductively coupled plasma-atomic emission spectrometer. The separation efficiency for $Zr^{4+}$ affinity column prepared by slurry packing was tested to phosphonated casein and dephosphonated casein. The resolution time (min) of the phosphonated casein was higher than that of dephosphated casein for $Zr^{4+}$ affinity polymeric microsphere by liquid chromatography. This $Zr^{4+}$ affinity column can be used for isolation of phosphonated casein from casein using liquid chromatography.

• Journal of Microbiology and Biotechnology
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• 제3권3호
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• pp.161-167
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• 1993

Immobilized metal ion affinity chromatography (IMAC) was used to separate various types of recombinant hirudins from the culture broth. The wild type hirudin exhibited a retention in Cu(II)-chelated affinity chromatgoraphy since it contained a single exposed histidine at position 51. To obtain a stronger retention on an IDA-Cu(II) column, the hirudin variants were genetically engineered to contain one or two histidine (s) more than the wild type. While the affinity of the variants for IDA-Cu(II) ligand increased in comparison to that of the wild type, the antithrombin activities reduced to a certain degree. Cu(II), Ni(II) and Zn(II) ions were applied separately to the metal chelate column to investigate ligand specificity with respect to protein retention. As a result, the Cu(II) chelated chromatography gave the best resolution for all the hirudins tested and appeared to be the only IMAC that could be used generally for the purification of hirudins with a decreasing pH gradient.

• KSBB Journal
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• 제13권2호
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• pp.125-132
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• 1998

Protein affinity membrane was prepared via the coating of chitosan gel on the porous flat polysulfone membrane surface, followed by the immobilization f the reactive dye (Cibacron Blue 3GA) to the chitonsan gel. The maximum protein binding capacity of affinity membrane was about 70${\mu}g/cm^2$ determined by the batch adsorption experiments of human serum albumin (HSA). Using module of this membrane, the characteristics of protein chromatography were investigated through the experiments of elution and frontal chromatography of HSA. This membrane module promises as a chromatography column, since it represented a lower pressure drop and a greater reproducibility. The protein separation ratio was significantly influenced by the flow rate of mobile phase and the injection quantity of HSA. The dynamic protein binding capacity of module decreased from the equilibrium binding capacity with increasing flow rate and approached the value of 15 - 20 ${\mu}g/cm^2$ for flow rates above 6 mL/min.

• 환경위생공학
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• 제12권2호
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• pp.59-64
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• 1997

For purification of a 70kDa toxic protein of mosquitocidal delta-endotoxin from B. thuringiensis strain H9B, immuno-affinity chromatography was performed. After separation of 70kDa toxic proteins from the delta-endotoxin of the strain H9B on SDS-PAGE, the 70kDa toxic protein was subcutaneously injected into rabbit for making a polyclonal antibody. A anti-70kDa toxic protein was purified by a column chromatography packed with protein A-sepharose 4B gels. The 70kDa toxic protein from delta-endotoxin of the strain H9B was also purified by an immuno-affinity chromatography packed with CNBr-activated sepharose 4B gels conjugated anti-70kDa toxic protein after elution with 1/10M citric acid-1/5M Na$_{2}$HPO$_{4}$ buffer(pH3.2) containing 0.5M NaCl. The 70kDa toxic protein was purified through only one step-separation system, was demonstrated by SDS-PAGE and immunoblot.

• Mass Spectrometry Letters
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• 제9권2호
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• pp.51-55
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• 2018

Capture of non-glycoproteins during lectin affinity chromatography is frequently observed, although it would seem to be anomalous. In actuality, lectin affinity chromatography works at post-translational modification (PTM) sites on a glycoprotein which is not involved in protein-protein interactions (PPIs). In this study, serial affinity column set (SACS) using lectins followed by proteomics methods was used to identify PPI mechanisms of captured proteins in human plasma. MetaCore, STRING, Ingenuity Pathway Analysis (IPA), and IntAct were individually used to elucidate the interactions of the identified abundant proteins and to obtain the corresponding interaction maps. The abundant non-glycoproteins were captured with the binding to the selected glycoproteins. Therefore, depletion process in sample pretreatment for abundant protein removal should be considered with more caution because it may lose precious disease-related low abundant proteins through PPIs of the removed abundant proteins in human plasma during the depletion process in biomarker discovery. Glycoproteins bearing specific glycans are frequently associated with cancer and can be specifically isolated by lectin affinity chromatography. Therefore, SACS using Lycopersicon esculentum lectin (LEL) can also be used to study disease interactomes.

• Journal of Animal Science and Technology
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• 제47권6호
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• pp.1025-1032
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• 2005

난백 단백질 중 ovotransferrin을 gel chromato- graphy와 heparin affinity chromatography를 통하여 분리 하였다. 1차 gel filtration의 경우에 샘플인젝션 후 65-70min(fraction No. 14) 이후에는 ovalbumin이 ovotransferrin과 혼입되어 분리되고 오히려 ovalbumin의 농도가 더 높은 분포를 보였다. 고순도의 ovotransferrin을 분리하기 위하여 다시 fraction No. 12-14을 농축한 뒤 gel filtration을 실시한 결과 ovotransferrin이 완전히 분리되지 않았는데 이는 gel filtration만의 반복을 통해서 순수한 ovotransferrin을 얻는 것이 비효과적임을 의미하는 것으로 판단된다. Ovotransferrin을 heparin affinity chromatography을 이용하여 분리한 경우 칼럼에 Fe2+를 고정시킨 후 50mM EDTA를 흘려 주었는데 ovalbumin이 5-10분경에 용출이 되었고 10-15분경에 ovalbumin과 ovotransferrin이 같이 용출되었다. 그 후에 50mM Phosphate buffer (pH 7.2, 0.15M salt)를 흘려주었는데 여전히 ovalbumin의 밴드가 보여 순수하지 않음을 확인하였다. Fe3+를 컬럼에 고정시킨 후 50mM EDTA를 흘려주었을 때 ovalbumin이 10-15분경에 용출이 되었고 15-20분경에 ovalbumin과 ovotransferrin이 같이 용출되었지만 50mM Phosphate buffer (pH 7.2, 0.15M salt free)를 흘려주었을 때 156-165분경에 ovalbumin이 혼입되지 않은 매우 순수한 ovotransferrin이 용출되는 것을 확인하였다. 위의 결과를 종합해볼 때 gel chromatography를 반복적으로 실시한 경우 보다는 heparin affinity chromatography를 이용하여 분리하고 컬럼에 Fe2+를 고정시킨 경우보다 Fe3+를 고정시켰을 때 더욱 순수한 ovotransferrin을 분리해낼 수 있었다.

• 생명과학회지
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• 제13권1호
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• pp.67-72
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• 2003

위염, 위궤양 및 위암의 원인 균인 Helicobacter pylori 가균체 표면에 다량 함유하며 주된 생존 인자이며 병원성 인자인 urease를 대장균에서 발현시키고 이 효소에 대한 항체 또는 기질과의 특이 상호 작용을 이용하여 두 단계의 간편한 방법에 의하여 정제하였다. 우선 anti-H. pylori Urease IgG-Sepharose column과 urea-Sepharose column을 각각 제조하고 DEAE-Sepharose 음이온 교환수지를 통하여 1차 정제한 시료를 각각 적용하고 제반 조건에서 용출시켰다. Anti-H. pylori urease IgG-Sepharose column의 경우에는 urease 시료가 너무 강력하게 결합함으로써 극단적인 pH조건에서만 용출이 가능함이 관찰되었으므로, 100 mM 탄산 완충액(pH 10.5)으로 최종 용출하였을 때 비교적 순수한 효소를 얻었으나, 비활성이 다소 감소된 것으로 나타났다. 한편, urea-Sepharose에 적용시킨 시료는 100 mM urea-HEB 완충액(pH 7.5)으로 비교적 용이하게 용출되어 비교적 높은 순도와 비활성의 urease 효소를 얻을 수 있었으나 이 경우에는 urease의 smaller subunit인 UreA peptide band의 강도가 다소 감소한 것이 관찰되었다.

• Bulletin of the Korean Chemical Society
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• 제18권6호
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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.