• 대한의생명과학회지
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• 제12권4호
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• pp.419-425
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• 2006

Phospholipase $C-{\gamma}1\;(PLC-{\gamma}1)$ is an important signaling molecule for cell proliferation and differentiation. $PLC-{\gamma}1$ contains two pleckstrin homology (PH) domains, which are responsible for protein-protein interaction and protein-lipid interaction. $PLC-{\gamma}1$ also has two Src homology (SH)2 domains and a SH3 domain, which are responsible for protein- protein interaction. To identity proteins that specifically binds to PH domain of $PLC-{\gamma}1$, we prepared and incubated the glutathione S-transferase(GST)-fused PH domains of $PLC-{\gamma}1$ with COS7 cell lysate. We found that 90 kDa protein specifically binds to PH domain of $PLC-{\gamma}1$. By matrix-assisted laser desorption ionization time of flight-mass spectrometry, the 90 kDa protein revealed to be heat shock protein (Hsp) $90{\beta}$. Hsp $90{\beta}$ is a molecular chaperone that stabilizes and facilitates the folding of proteins that are involved in cell signaling, including receptors for steroids hormones and a variety of protein kinases. To know whether Hsp $90{\beta}$ affects on $PLC-{\gamma}1$ activity, we performed $PIP_2$ hydrolyzing activity of $PLC-{\gamma}1$ in the presence of purified Hsp $90{\beta}$ in vitro. Our results show that the Hsp $90{\beta}$ dose-dependently inhibits the enzymatic activity of $PLC-{\gamma}1$ and further suggest that Hsp $90{\beta}$ regulates cell growth and differentiation via regulation of $PLC-{\gamma}1$ activity.

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.474-481
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• 1996

Since the commercially available rabbit anti-cyclin D3, generated from c-terminal 16 amino acid residues which are common to human and murine cyclin D3, is highly cross-reactive with many other cellular proteins of mouse, a new rabbit polyclonal anti-cyclin D3 has been raised by using murine cyclin D3 protein expressed at a high level in Escherichia coli as the immunogen. To express murine cyclin D3 protein in E. coli, the cyclin D3 cDNA fragment encoding c-terminal 236 amino acid residues obtained by polymerase chain reaction (PCR) was inserted into the NcoI/BamHI site of protein expression vector, pET 3d. Molecular mass of the cyclin D3 overexpressed in the presence of IPTG (Isopropyl $\beta$-D-thiogalactopyranoside) was approximately 26 kDa as calculated from the reading frame on the DNA sequence, and the protein was insoluble and mainly localized in the inclusion bodies that could be easily purified from the other cellular soluble proteins. When renaturation was performed following denaturation of the insoluble cyclin D3 protein in the inclusion bodies using guanidine hydrochloride, 4.4 mg of soluble form of cyclin D3 protein was produced from the transformant cultured in 100ml of LB media under the optimum conditions. Four-hundred micrograms of the soluble form of cyclin D3 protein was used for each immunization of a rabbit. When the antiserum obtained 2 weeks after tertiary immunization was applied to Western blot analysis, it was able to detect 33 kDa cyclin D3 protein in both murine lymphoma cell line BW5147.G.1.4 and human Jurkat T cells at 3,000-fold dilution with higher specificity to murine cyclin D3, demonstrating that the new rabbit polyclonal anti-murine cyclin D3 generated against c-terminal 236 amino acid residues more specifically recognizes murine cyclin D3 protein than does the commercially available rabbit polyclonal antibody raised against c-terminal 16 amino acids residues.

• 한국식품과학회지
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• 제3권2호
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• pp.101-104
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• 1971

Frankel-Conrat 와 Cooper에 의해 개발된 쉽고 빠르며 능률적으로 단백질 함량 및 질을 결정할 수 있는 염색법을 고단백 품종의 선발에 이용할 수 있는지의 여부를 알기 위하여 최근 고단백 수도품종으로 육종되고 있는 IR-667 계통에 적용하여 단백질, BAA, 및 라이신 함량과의 관계를 비교 검토하여 다음과 같은 결론을 얻었다. DBA-protein의 관계는 고도의 상관관계를 보였으며 단백질 함량을 생체중으로 나타냈을 때 $-0.975^{**}$, 건물중으로 나타냈을 때 $-0.955^{**}$의 상관계수를 보였다. DBA-lysine 및 DBA-BAA의 상관관계는 모두 고도의 상관관계를 보였으며 비슷한 상관계수를 보였다. DBA-lysine의 관계는 DBA-protein 의 관계보다는 다소 낮으나 protein-lysine과의 관계보다는 높은 상관관계를 나타내었다.

• KSBB Journal
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• 제30권6호
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• pp.296-301
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• 2015

Eggshell-based biocomposites have become attractive due to their exquisite nanostructure and biological properties, which are mainly composed of highly organized calcium carbonate crystals controlled by organic macromolecules such as proteins and polysaccharides. Here, we designed the recombinant fusion protein of a putative eggshell matrix protein named as GG1234 and a fluorescent reporter protein of DsRed. The protein was successfully over-expressed in E. coli and purified by Ni-NTA affinity chromatography. In vitro calcium carbonate crystallization was conducted in the presence of the fusion protein, and morphological change was investigated. The protein inhibited the calcite growth in vitro, and spherical calcium carbonate micro-particles with the diameter of about $20-30{\mu}m$ were obtained. We expect that this study would be helpful for better understanding of eggshell-based biomineralization.

• 한국자기공명학회논문지
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• 제21권1호
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• pp.13-19
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• 2017

Human sterol ${\Delta}8-{\Delta}7$ isomerase, commonly known as emopamil binding protein (EBP), is an essential protein in the cholesterol-synthetic pathway, and mutations of this protein are critically associated with human diseases such as Conradi-Hunermann-Happle or male EBP disorder with neurological defects syndrome. Due to such a clinical importance, EBP has been intensively investigated and some important features have been reported. EBP is a tetra-spanning membrane protein, of which $2^{nd}$, $3^{rd}$, and $4^{th}$ membrane-spanning ${\alpha}$ helices play an important role in its enzymatic function. However, detailed structural feature at atomic resolution has not yet been elucidated, due to characteristic difficulties in dealing with membrane protein. Here, we over-expressed EBP using Escherichia coli and performed detergent screening to find suitable membrane mimetics for structural studies of the protein by NMR. As results, DPC and LMPG could be evaluated as the most favorable detergents to acquire promising NMR spectra for structural study of EBP.

• Animal cells and systems
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• 제2권3호
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• pp.355-359
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• 1998

Among the proteins secreted from Lactobacillus acidophilus KCTC 3151, a 36 kDA and 24 kDa protein, whose amounts were relatively abundant, were purified and their N-terminal amino acid sequences determined. The N-terminal amino acid sequence of 36 kDa protein exhibited high homology with thymidine phosphorylase and glyceraldehyde-3-phosphate dehydrogenase. The N-terminal amino acid sequence of the 24 kDa protein did not show significant homology with proteins in Protein Data Base nor Gene Bank. Nucleotide sequence of the gene encoding 36 kDa protein indicates that the protein possesses the domains for a-helical, phosphate binding and pyrimidine binding sites, which are also shown in thymidine phosphorylases. Also, the protein contains conserved domains of dehydrogenase II and III. However, the activity of thymidine phosphorylase or glyceraldehyde-3-puospnate dehydrogenase could not be detected in the purified fractions of the 36 kDa protein.

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.468-473
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• 1996

The genome of tomato spotted wilt virus (TSWV) is composed of three RNA segments, S, M, and L RNA and the 5.0 kb M RNA encodes two glycoproteins Gl, G2 and NSm protein of unknown function. In an effort to investigate the function of the NSm protein, antibody was raised against NSm fusion protein overexpressed in Escherichia coli. This antibody was used to detect the NSm protein by using western blot analysis and electron microscopic observation after immunogold labelling. For the cloning of the NSm gene, total RNA extracted from a TSWV infected plant was used for cDNA synthesis and polymerase chain reaction (PCR) instead of going through time-consuming virus purification. A protein band specifically reacting to the NSm antibody was detected from TSWV inoculated plants. The NSm protein was detected in the cell wall fraction and in pellet from low speed centrifugation when the infected plant tissue was fractionated into 4 fractions. In the immuno-electron microscopic observation, gold particles were found around the plasmodesmata of infected plant tissue. These results suggest that the NSm protein of TSWV plays some role in cell-to-cell movement of this virus.

• BMB Reports
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• 제28권6호
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• pp.504-508
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• 1995

The electrophoretic patterns of plasma membrane surface proteins of normal rat liver cells and rat hepatomas were compared in 10% non-denaturing and 7-15% gradient non-denaturing gel. Chemical carcinogens, 2-Me DAB (2-methyl-4-dimethylaminoazobenzene) and DENA (diethylnitrosamine), were used to induce hepatoma in rats. One protein which disappeared in hepatoma was identified in normal rat liver by non-denaturing gel electrophoresis. Rabbit antisera were raised against this specific protein, and the protein was purified by Sephacryl S-200 column and immunoaffinity chromatography using the purified antibody. The purified protein showed two bands of molecular weights approximately 50 $kD_{\alpha}$ and 52 $kD_{\alpha}$ by SDS-polyacrylamide gel electrophoresis, which reacted specifically with the antibody. However only one band was observed in non-denaturing gel and also in isoelectric focusing with a pI value of 6.6. This study showed the existence of an unique protein on the plasma membrane surface of normal rat liver cells which disappeared in rat hepatomas induced by chemical carcinogens.

• Journal of Nutrition and Health
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• 제6권2호
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• pp.1-16
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• 1973

Some effects of dietary conditions on the metabolism of nucleic acid and protein in organs of the Albino Rats have been studied. The young rats to be examined were fed on the control diet and the diets deprived of one component among protein, carbohydrate, and lipid, such as protein free diet (PF: 432 kcal/100g) carbohydrate free diet (CF: 432kcal/100g), and lipid free diet (LF: 392kcal/100g) for three, seven, and fifteen days, respectively. The contents of DNA and RNA in the liver and the brain, and also those of protein-nitrogen(PN) and nonprotein-nitrogen (NPN) in the live, the brain, and the serum have been measured. The results are as followe: 1. The contents of DNA per gram of liver were increased by feeding on protein free diet. It is concluded that the critical factor for the result is not the increase in the rates of DNA syntheses, but the decrease in the turnover rates of DNA. 2. The metabolism of DNA in the liver showed the normal status by feeding on carbohydrate free diet. On the other hand, the rates of DNA syntheses were increased by feeding on lipid free diet. 3. The rates of DNA syntheses in the brain were decreased by feeding on the unbalanced diet, such as protein free, carbohydrete free, and lipid free diet. 4. In the liver and the brain, the rates of DNA syntheses were decreased by feeding on protein free diet. But the rates showed the normal status by feeding on the carbohydrate free diet, and also showed the similar metabolism to that in the case of the control group by feeding on lipid free diet. 5. In the liver, the rates of protein syntheses were decreased, whereas the contents of nonprotein-nitrogen were increased by feeding on protein free diet. 6. In the liver and the brain, the protein syntheses showed the more increasing rates than the rates in the case of the control diet by feeding on lipid free diet. 7. In the serum, the contents of protein did not change in a short period, also the insufficient feeling on protein was examined. It is clear that in the liver the rates of protein syntheses are decreased and the rates of protein catabolism are increased, since the rates of nucleic acid syntheses are decreased by feeding on the protein free diet. On the other hand, it is considered that in the brain the turnover rates of protein does not have correlation with the rates of nucleic acid syntheses, also these are decreased by feeding on protein free diet. And also it is believed that the phenomena of homeostasis for carrying the normal metabolism of nucleic acid and protein in the liver and the brain are operated in a short period as possible, by feeding on carbohydrate free and lipid free diets.

• Archives of Pharmacal Research
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• 제21권6호
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• pp.712-717
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• 1998

The complete nucleotide sequence of the cDNA clone encoding rat skeletal muscle myosin- binding protein H (MyBP-H) was determined and amino acid sequence was deduced from the nucleotide sequence (GenBank accession number AF077338). The full-length cDNA of 1782 base pairs(bp) contains a single open reading frame of 1454 bp encoding a rat MyBP-H protein of the predicted molecular mass 52.7kDa and includes the common consensus 1CA__TG' protein binding motif. The cDNA sequence of rat MyBP-H show 92%, 84% and 41% homology with those of mouse, human and chicken, respectively. The protein contains tandem internal motifs array (-FN III-Ig C2-FN III- Ig C2-) in the C-terminal region which resembles to the immunoglobulin superfamily C2 and fibronectin type III motifs. The amino acid sequence of the C-terminal Ig C2 was highly conserved among MyBPs family and other thick filament binding proteins, suggesting that the C-terminal Ig C2 might play an important role in its function. All proteins belonging to MyBP-H member contains `RKPS` sequence which is assumed to be cAMP- and cGMP-dependent protein kinase A phosphorylation site. Computer analysis of the primary sequence of rat MyBP-H predicted 11 protein kinase C (PKC)phosphorylation site, 7 casein kinase II (CK2) phosphorylation site and 4N-myristoylation site.