• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1557-1564
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• 2009

Human CDX2 is known as a caudal-related homeodomain transcription factor that is expressed in the intestinal epithelium and is important in differentiation and maintenance of the intestinal epithelial cells. The caudal-related homeobox proteins bind DNA according to a helix-turn-helix structure, thereby increasing the structural stability of DNA. A cancer-tumor suppressor role for Cdx2 has been shown by a decrease in the level of the expression of Cdx2 in colorectal cancer, but the mechanism of transcriptional regulation has not been examined at the molecular level. We developed a large-scale system for expression of the recombinant, novel CDX2, in Escherichia coli. A highly purified and soluble CDX2 protein was obtained in E. coli strain BL21(DE3)RIL and a hexahistidine fusion system using Ni-NTA affinity column, anion exchange, and gel filtration chromatographies. The identity and secondary structure of the purified CDX2 protein were confirmed by MALDI-TOF MS, Western blot, and a circular dichroism analyses. In addition, we studied the DNA-binding activity of recombinant CDX2 by ELISA experiment and isolated human CDX2-binding proteins derived from rat cells by an immobilized GST-fusion method. Three CDX2-binding proteins were found in the gastric tissue, and those proteins were identified to the homeobox protein Hox-D8, LIM homeobox protein 6, and SMC1L1 protein.

• 대한의생명과학회지
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• 제2권2호
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• pp.231-240
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• 1996

인체 혈액 응고 9인자는 간에서 생성되며 461개의 아미노산으로 구성된 당 단백질이다. 따라서 인체 혈액 응고 9인자 cDNA를 찾기 위해 태아의 간(fetal liver) cDNA library를 PCR(Polymerase Chain reaction) 방법으로 screening하였으며, 그 결과 ATG개시 코돈으로부터 TAA종료 코돈까지 포함하는 1.4 kb의 9인자 cDNA를 찾았다. 또한 클론된 9인자 cDNA를 박테리아에서 발현시키기 위해 박테리아 발현 벡터인 pGEX-2T 플라스미드에 클로닝하므로써 pGEX-F9 플라스미드를 제조하였다. pGEX-F9로 형질전환된 E. coli에서 PGEX-F9의 발현을 유도하면 73 kDa 크기의 GST-factor9 융합 단백질이 다량생성되며 , 이 단백질이 혈액 응고 9인자 단백질을 함유하는 융합 단잭질임을 혈액 응고 9인자 항체를 이용한 Western blot으로 입증하였다. E. coli에서 발현된 GST-factor 9 융합 단백질은 전체 단백질의 약 20%를 차지하며 GST agarose bead를 이용한 one step purificarion 방법을 통해 GST-factor9 융합 단백질을 쉽게 분리 할 수 있다.

• BMB Reports
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• 제29권3호
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• pp.236-240
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• 1996

A 23-kDa molecular mass of antioxidant protein was purified from human liver. This protein exhibited the preventive effect against the inactivation of glutamine synthetase by a metal-catalyzed oxidation system. This antioxidant activity was supported by a thiol-reducing equivalent such as dithiothreitol in a similar manner to that of the 25-kDa thiol-specific antioxidant protein (TSA) from human red blood cells (HR). However, a thioredoxin-linked peroxidase activity of thiol-specific antioxidant protein of human liver (HLTSA) (0.91 ${\mu}mol/min/nmol$ of HLTSA) was much lower than that of thiol-specific antioxidant protein of human red blood cells (HRTSA) (16.4 ${\mu}mol/min/nmol$ of HRTSA). This HLTSA is also immnologically distinct from HRTSA Amino acid sequences of the three tryptic peptides (P1, P2, P3) of HLTSA were found to be completely homologous to segments of the known Mer5-like protein, which belongs to the known TSA family.

• BMB Reports
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• 제42권11호
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• pp.697-704
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• 2009

Membrane proteins play important roles in the biology of the cell, including intercellular communication and molecular transport. Their well-established importance notwithstanding, the high-resolution structures of membrane proteins remain elusive due to difficulties in protein expression, purification and crystallization. Thus, accurate prediction of membrane protein topology can increase the understanding of membrane protein function. Here, we provide a brief review of the diverse computational methods for predicting membrane protein structure and function, including recent progress and essential bioinformatics tools. Our hope is that this review will be instructive to users studying membrane protein biology in their choice of appropriate bioinformatics methods.

• Journal of Microbiology and Biotechnology
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• 제8권5호
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• pp.466-470
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• 1998

The basic and optimum conditions for the extraction of peroxidase from Chinese cabbage root applying the reverse micelle system were investigated. In order to purify Peroxidase (POX) from crude extract of Chinese cabbage roots, isooctane containing 110 mM Aerosol OT (AOT) was well mixed with the same volume of crude extracts containing 50 mM NaCl and 30 mM Tris-HCI buffer of pH 8.0. After centrifugation, AOT reverse micelle containing the isooctane phase were mixed with 80 mM Tris-HCI buffer at pH 7.0 containing 1 M KCl. From these operations, POX was purified 20-fold with a 60% yield. For further purification, DEAE-Toyopearl column chromatography was applied, and it showed a single protein band on SDS-polyacrylamide gel electrophoresis. The resulting POX showed 93-fold purification with a 40% yield.

• Journal of Microbiology and Biotechnology
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• 제19권4호
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• pp.416-423
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• 2009

Hepatitis B core antigen(HBcAg) is an important serological marker used in the diagnosis of hepatitis B virus(HBV) infections. In the current study, a fast and efficient preparative purification protocol for truncated HBcAg from Escherichia coli disruptate was developed. The recombinant HBcAg was first captured by anion exchange expanded bed adsorption chromatography integrated with a cell disruption process. This online capture process has shortened the process time and eliminated the "hold-up" period that may be detrimental to the quality of target protein. The eluted product from the expanded bed adsorption chromatography was subsequently purified using size-exclusion chromatography. The results showed that this novel purification protocol achieved a recovery yield of 45.1% with a product purity of 88.2%, which corresponds to a purification factor of 4.5. The recovered HBcAg is still biologically active as shown by ELISA test.

• 생명과학회지
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• 제16권3호
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• pp.387-395
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• 2006

유도된 집파리유통 hemolymph중에서 Candida albicans의 3가지 anti-fungal peptides를 분리하였다. 3개 anti-fungal peptides는 분자량이 4-16 kDa 사이의 분명한 구별이 있을 뿐만 아니라, 각 peptide는 anti-fungal peptides작용이 있었다. 이들 peptide의 공통 특징은 모두 열을 받은 뒤 활성이 변하지 않는 비교적 강한 내열성을 보여주었다.

• 한국미생물·생명공학회지
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• 제20권4호
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• pp.401-408
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• 1992

Cholesterol oxidase를 생산하는 균주를 토양으로 분리하고 이를 배양하여 효소를 생산한 후 정제하여 그 특성을 조사하였다. 본 효소는 CH2 concentrator에 의한 농축, DEAE-cellulose chromatography, Superose 12 column FPLC로 정제하였고, 비활성이 약 31배나 증가하였다. 이 효소는 $50^{\circ}C$, pH 6.0에서 최대 활성을 나타냈고 30-$45^{\circ}C$ 범위에서는 안정하였다. pH에 대한 안정성은 pH 6.0-11.0까지 광범위한 안정성을 보였으며, Km 값은 cholesterol에 대해서 $3.65{\times}10^{-3}$M이며 분자량은 59,500 dalton으로 추정 된다. 또한 $Ca^{2+}$, $Ba^{2+}$, $Fe^{2+}$, Brij 35에 의해서는 효소의 활성이 저해되지 않았지만, $Hg^{2+}$, $Ag^{2+}$ 및 SDS에 의해서는 저해되는 것으로 나타났다.

• 한국식품영양과학회지
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• 제27권5호
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• pp.808-812
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• 1998

Melania snail(Semisulcopira bensoni) is used as ingredient in Korean traditional soup and nutritional foods. Generally, lipoxygenase in several food products may produce off-flavors during their processing and storage. Therefore, the inactivation of lipoxygenase is required to make the better extracts from Melania sanil. Also, the quality on freshness of Melania snail may be evaluated by lipoxygenase activity. The lipoxygenae activity was the highest at 40~60% saturation among several concentrations in salting-ouot saturated solution of ammonium sulfate. The partial purification of lipoxygenase was successfully obtained by Sephacryl S-200 gel chromatography. The first peak among three peaks for protein determination showed the highest activity of lipoxygenase in 13~16 fractions among 100 fractions. The highest peak of lipoxygenase activity by ion exchange chromatography was shown at 0.1M NaCl. In the purification step, the specific activity was 20.8U/mg and activity yield was 19.8%. The optimum pH and temperature were pH6.0~8.0 and 3$0^{\circ}C$, respectively. Molecular weight of the lipoxygenase was estimated about 35kDa by SDS-PAGE.

• 한국자기공명학회논문지
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• 제20권1호
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• pp.22-26
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• 2016

FANCJ is a DNA helicase which contributes genome stability by resolving G-quadruplex DNA from 5' to 3' direction. In addition to main ATPase helicase core, FANCJ has the protein binding region at its C-terminal part. BRCA1 and BLM are the binding partner of FANCJ and these protein-protein interactions contribute genomic stability and the proper response to replication stress. As the first attempt for studying FANCJ-BLM interaction, we prepared BLM binding region of FANCJ and characterized with CD and NMR spectroscopy. FANCJ (881-941) with N-ter 6xHis was purified as the oligomer. Secondary structure prediction based on CD data revealed that FANCJ (881-941) composed with ${\beta}$ sheet, turn and coils.$^1H-^{15}N$ HSQC spectra showed nonhomogeneous peak intensities with less number of peaks comparing than the number of amino acids in the construct. It indicated that optimization should be necessary for detailed further structural studies.