• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1992년도 제1회 신약개발 연구발표회 초록집
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• pp.42-42
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• 1992

ras oncogene은 암조직이나 transformed human cell line에서 거의 공통적으로 발견되는 oncogene으로서 그 product인 p2l 단백질은 C-terminal 25개의 아미노산 외에는 거의 동일한 배열을 가지고 있는 매우 conservative한 단백질이며 C-terminal cysteine이 carboxy methylation되어 있고 또한 palmitic acid와 같은 long chain fatty acid도 결합되어 있다. 보고된 바에 의하면 p21 protein의 palmitation은 ras protein의 세포막에 대한 친화력을 유지시키며 이와 같은 친화력은 cell transforming activity의 기본요건으로 알려져 있다. 이와 같은 관점에서 볼때 p21 단백질의 C-terminal processing현상을 new drug target으로, 즉 p2l 단백질의 C-terminal processing을 억제하므로서 cell transforming activity를 저해 할 수 있을 것이므로 생체내에 존재하는 p21 단백질 C-terminal processing 억제제의 identification 및 purification은 항암제 연구와 밀접한 관계가 있다. 구체적으로 farnesyl-protein transferase inhibitor 혹은 carboxyl methyl inhibitor의 identification 및 purification은 이같은 목적을 달성 할 수 있는 가능성이 크다.

• BMB Reports
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• 제41권12호
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• pp.881-885
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• 2008

Protein phosphatase 1 consists of a secondary structure arrangement, conserved in the serine/threonine protein phosphatase gene family, flanked by nonconserved N-terminal and C-terminal domains. The deletion mutant of PP1 with the 8 nonconserved N-terminal residues removed was designated PP1-(9-330). PP1-(9-330) had a higher activity and affinity than PP1 when assayed against four different substrates, and it also demonstrated a 6-fold higher sensitivity to the inhibitor okadaic acid. This suggested that the N-terminal domain suppresed the activity of PP1 and interfered with its inhibition by okadaic acid. The ANS fluorescence intensity of PP1-(9-330) was greater than that of PP1, which implies that the hydrophobic groove running from active site in the truncated PP1 was more hydrophobic than in PP1. Our findings provide evidence that the nonconserved N-terminus of PP1 functions as an important regulatory domain that influences the active site and its pertinent properties.

• 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.

• 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.

• Archives of Pharmacal Research
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• 제21권3호
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• pp.291-297
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• 1998

Cop protein has been overexpressed in Escherichia coli using a T7 RNA polymerase system. Purification to apparent homogeneity was achieved by the sequential chromatography on ion exchange, affinity chromatography, and reverse phase high performance liquid chromatography system. The molecular weight of the purified Cop was estimated as 6.1 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). But the molecular mass of the native state Cop was shown to be 19 kDa by an analytical high performance size exclusion chromatography, suggesting a trimer-like structure in 50 mM Tris-HCI buffer (pH 7.5) containing 100 mM NaCl. Cop protein Was calculated to contain $39.1% {\alpha}-helix, 16.8% {\beta}-sheet$, 17.4% turn, and 26.8% random structure. The DNA binding property of Cop protein expressed in E. coli Was preserved during the expression and purification process. The isoelectric point of Cop was determined to be 9.0. The results of amino acid composition analysis and N-terminal amino acid sequencing of Cop showed that it has the same amino acid composition and N-terminal amino acid sequence as those deduced from its DNA sequence analysis, except for the partial removal of N-terminal methionine residue by methionyl-aminopeptidase in E. coli.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.727-730
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• 2001

Xanthomonas oryzae #5로부터 클로닝 된 CFTase 의 functional domain의 분석을 위해 CFTase의 recombinant deletion mutant를 구성하고, recombinant protein을 분리, 정제하였다. 분리, 정제한 recombinant protein의 활성을 측정한 결과 C-terminal이 deletion 된 mutant는 cyclization 반응이 소실 되었다. 이와 같은 결과로부터 CFTase의 C-terminal 은 cyclization 반응의 중요한 functional domain 이다.

• 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.

• 대한수의학회지
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• 제40권1호
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• pp.86-93
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• 2000

Paratuberculosis (Johne's disease), a chronic enteritis produced by Mycobacterium paratuberculosis, affects a large proportion of ruminants in all continents and causes important economic losses. The identification of well-characterized and species-specific components of M paratuberculosis would provide the means to improve the specificity and sensitivity of immunodiagnostic assays for Johne's disease. The aims of this study were to express the recombinant C-terminal of 34kDa protein (rC34P) of M paratuberculosis in E coli and to investigate the effectiveness of this protein in detecting antibodies to the native protein in sera from paratuberculosis infected cattle. The C-terminal of the gene encoding the 34kDa protein was amplified by polymerase chain reaction from the chromosomal DNA of M paratuberculosis (ATCC 19698) and cloned into vector pGEX-4T-2. Then, cloned plasmid was transformed into E coli DH5${\alpha}$ and the rC34P was overexpressed. The rC34P was purified by affinity chromatography and gel filtration. The rC34P was examined antigenicity by Western blot. The rC34P was reactive with culture positive bovine serum and hyperimmune rabbit anti-M paratuberculosis serum but was not reactive with culture negative bovine serum and tuberculin positive bovine serum in Western blot. In conclusion, the rC34P produced in this study is expected as a useful candidate for antigen in serological diagnosis of Johne's disease.

• Bulletin of the Korean Chemical Society
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• 제32권10호
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• pp.3553-3554
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• 2011

• Biomolecules & Therapeutics
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• 제4권1호
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• pp.97-102
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• 1996

To investigate whether human $\beta$$_2$-adrenergic receptor devoid of the C-terminal two transmembrane helices retain its ligand binding activity and specificity, 5'780-bp DNA fragment of the receptor gene which encodes amino acid 1-260 of human $\beta$$_2$-adrenergic receptor was subcloned into the bacterial fusion protein expression vector and expressed as a form of glutathione-S-transferase (GST) fusion protein in E. coli DH5$\alpha$. The receptor fusion protein was expressed as a membrane bound form which was verified by SDS-PAGE and Western blot. The fusion protein expressed in this study specifically bound $\beta$-adrenergic receptor ligand [$^3$H] Dihydroalprenolol. In saturation ligand binding assay, the $K_{d}$ value was 7.6 nM which was similar to that of intact $\beta$$_2$-adrenergic receptor in normal animal tissue ( $K_{d}$=1~2 nM) and the $B_{max}$ value was 266 fmol/mg membrane protein. In competition binding assay, the order of binding affinity of various adrenergic receptor agonists to the fusion protein was isoproterenol》epinephrine norepinephrine, which was similar to that of intact receptor in normal animal tissue. These results suggest that N-terminal five transmembrane helices of the $\beta$$_2$-adrenergic receptor be sufficient to determine the ligand binding activity and specificity, irrespective of the presence or absence of the C-terminal two transmembrane helices.s.s.s.