• BMB Reports
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• 제32권1호
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• pp.12-19
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• 1999

Gaegurin 4 (GGN4), a broad-spectrum antibiotic, is a 37-amino acid peptide isolated from the Korean frog, Rana rugosa. In this study, we have chemically synthesized and purified GGN4 analogues where the C-terminal portion is truncated and/or substituted with tryptophan. These peptides show significantly different biological activities depending on the location of tryptophan and the number of amino acids truncated from the C-terminal end. While deletion of 9 amino acids from the C-terminal seems to be marginally tolerable in maintaining the antimicrobial activity, further deletion of up to 14 amino acid residues decreases the potency by more than 60-fold towards Gram-positive, and 10-fold towards Gram-negative, bacteria. Surprisingly, the reduced activity of the shorter peptide can be completely restored by a single substitution of aspartic acid 16 to tryptophan 16 (D16W). Also, the truncation seems to decrease the specificity of antibiotic activity more towards Gram-positive than towards Gram-negative bacteria studied. These data suggest a partial role of the C-terminal region in determining the binding specificity and the activity of peptides upon binding to their target cell membranes.

• 약학회지
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• 제49권3호
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• pp.198-204
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• 2005

Human foamy virus (HFV) integrase mediates integration of viral c-DNA into cellular DNA. In this process, HFV integrase recognizes its own viral DNA specifically and catalyzes insertion of viral c-DNA. In order to study catalytic domains and residues, three deletion mutants and two point mutants of HFV integrase were constructed and analyzed with respect to enzymatic activities. The C-terminal deletion mutant showed decreased enzymatic activities while the N-terminal deletion mutant lost the activities completely, indicating that the N-terminal domain is more important than the C-terminal domain in enzymatic reaction. The point mutants, in which an aspartic acid at the 164th position or a glutamic acid at the 200th position of the HFV integrase protein was changed to an alanine, lost the enzymatic activities completely. However, they were well complemented with other defective deletion mutants to recover enzymatic activities partially. Therefore, these results suggest that the aspartic acid and glutamic acid at the respective 164th and 200th positions are catalytic residues for enzymatic reaction.

• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1477-1480
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• 2006

The 63-amino-acid-encoding afsR2 is a global antibiotics-stimulating regulatory gene identified from the chromosome of Streptomyces lividans. To dissect a putative functional domain in afsR2, several afsR2-derivative deletion constructs were generated and screened for the loss of actinorhodin-stimulating capability. The afsR2-derivative construct missing a 50-bp C-terminal region significantly lost its actinorhodin-stimulating capability in S. lividans. In addition, site-directed mutagenesis on amino acid positions of #57-#61 in a 50-bp C-terminal region, some of which are conserved among known Sigma 70 family proteins, significantly changed the AfsR2's activity. These results imply that the C-terminal region of AfsR2 is functionally important for antibiotics-stimulating capability and the regulatory mechanism might be somehow related to the sigma-like domain present in the C-terminal of AfsR2.

• Journal of Microbiology and Biotechnology
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• 제15권1호
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• pp.141-146
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• 2005

A new system for displaying proteins on the surface of Escherichia coli was developed using the Salmonella typhimurium outer membrane protein C (OmpC) as an anchoring motif. The C-terminal deletionfusion strategy was developed to fuse the polyhistidine peptides and green fluorescent protein (GFP) to the Cterminal of the truncated functional portion of OmpC. The polyhistidine peptides of up to 243 amino acids could besuccessfully displayed on the E. coli cell surface, which allowed recombinant E. coli to adsorb up to 34.2 μmol of Cd2+ per gram dry cell weight. The GFP could also be successfully displayed on the E. coli cell surface. These results suggest that the C-terminal deletion-fusion strategy employing the S. typhimurium OmpC as an anchoring motif provides a new efficient way for the display of large proteins on the surface of E. coli.

• BMB Reports
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• 제43권12호
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• pp.801-806
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• 2010

The existence of glycine residues in long-chain scorpion toxins has been well documented. However, their role as analgesics has not been evaluated. To address this issue, we investigated the functional role of glycines in the C-terminal end of Chinese-scorpion toxin from Buthus martensii Karsch (BmK AGP-SYPU2) using site-directed mutagenesis and analgesic activity assays. Recombinant BmK AGP-SYPU2 and its mutants were efficiently expressed in E. coli and purified to homogeneity using immobilized metal ion affinity chromatography (IMAC) and cation exchange chromatography. The mouse-twisting test was used to detect the analgesic activity of BmK AGP-SYPU2 and its mutants. As a result, we identified glycines at the C-terminal end that, when altered, significantly affected analgesic activity. Also, Mut6566 was significantly decreased compared to BmK AGP-SYPU2. These data indicate that the glycines at the C-terminal end are important for the analgesic activity of BmK AGP-SYPU2.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2002년도 창립10주년기념 및 국립독성연구원 의약품동등성평가부서 신설기념 국재학술대회:생물학적 동등성과 의약품 개발 전략을 위한 국제심포지움
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• pp.236-236
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• 2002

Pyruvate dehydrogenase phosphatase (PDP) is a mitochondrial protein serine/threonine phosphatase that catalyzes the dephosphorylation and concomitant reactivation of the pyruvate dehydrogenase componant of the pyruvate dehydrogenase complex (PDC). PDP consists of a Mg$\^$+2/ -dependent and Ca$\^$+2)-stimulated catalytic subunit (PDPc) of Mr 52,600 and a FAD-containing regulatory subunit (PDPr) of Mr 95.600. Catalytic subunit of pyruvate dehydrogenase phosphatase (PDPc) has been suggested to have three major functional domains such as dihydrolipoamide acetyltransferase(E$_2$)-binding domain, regulatory subunit of PDP(PDPr)-binding domain, and calcium-binding domain. In order to identify functional domains, recombinant catalytic subunit of pyruvate dehydrogenase phosphatase (rPDPc) was expressed in E. coli JM101 and purified to near homogeneity using the unique property of PDPc: PDPm binds to the inner lipoyl domain (L$_2$) of E$_2$ of pyruvate dehydrogenase complex (PDC) in the presence of Ca$\^$+2/, not under EGTA. PDPc was limited-proteolysed by trypsin, chymotrypsin, Arg-C, and elastase at pH7.0 and 30$^{\circ}C$ and N-terminal analysis of the fragment was done. Chymotrypsin, trypsin, and elastase made two major framents: N-terminal large fragment, approx. 50kD and C-terminal small fragment, approx. 0 kDa. Arg-C made three major fragments: N-terminal fragment, approx. 35 kD, and central fragment, approx. 15 kD, and C-terminal fragment, approx. 10 kD. This study strongly suggest that PDPc consists of three major functional domains. However, further study should be necessary to identify the functional role.

• The Korean Journal of Physiology and Pharmacology
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• 제5권6호
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• pp.495-501
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• 2001

We have cloned the mouse neurotensin/neuromedin N (NT/N) gene from the murine mast cell line Cl.MC/C57.1 for the first time. The murine NT/N cDNA clone consisted of 765 nucleotides and coded for 169 peptide residues with an N-terminal signal peptide, and the C-terminal region contained of one copy of neurotensin (NT) and one copy of neuromedin N (NN). Total of four Lys-Arg dibasic motifs were present; one each at the middle of the open reading frame, at the N-terminal of NN, at the C-terminal of NT, and between NN and NT. Amino acid sequence analysis of the mouse NT/N revealed 90% homology to that of the rat NT/N gene. NT/N is expressed in murine mast cell lines (Cl.MC/C57.1 and P815), but not in murine bone marrow-derived mast cells (BMMCs), murine macrophage cell line (RAW 264.7), nor in murine T cell line (EL-4). NT/N mRNA in C1.MC/C57.1 is highly inducible by IgE cross-linking, phorbol myristate acetate, neurotensin, and substance P. Following the treatment of demethylating agent, 5-azacytidine (5-azaC), the NT/N gene was induced in BMMCs in response to IgE cross-linking. 5-azaC-treated BMMCs did not express the NT/N gene without additional stimuli. These findings suggested that the regulation of NT/N gene expression was dependent on the effects of not only gene methylation but also enhancer and/or repressor proteins acting on the NT/N promoter.

• BMB Reports
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• 제46권1호
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• pp.53-58
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• 2013

We constructed deletion mutants and seven point mutants by polymerase chain reaction to investigate the specificity of feline foamy virus integrase functional domains. Complementation reactions were performed for three enzymatic activities such as 3'-end processing, strand transfer, and disintegration. The complementation reactions with deletion mutants showed several activities for 3'-end processing and strand transfer. The conserved central domain and the combination of the N-terminal or C-terminal domains increased disintegration activity significantly. In the complementation reactions between deletion and point mutants, the combination between D107V and deletion mutants revealed 3'-end processing activities, but the combination with others did not have any activity, including strand transfer activities. Disintegration activity increased evenly, except the combination with glutamic acid 200. These results suggest that an intact central domain mediates enzymatic activities but fails to show these activities in the absence of the N-terminal or C-terminal domains.

• 한국발생생물학회지:발생과생식
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• 제26권1호
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• pp.1-12
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• 2022

This study aimed to investigate the signal transduction of phosphorylation sites at the carboxyl (C)-terminal region of equine luteinizing hormone/chorionic gonadotropin receptor (eLH/CGR). The eLH/CGR has a large extracellular domain of glycoprotein hormone receptors within the G protein-coupled receptors. We constructed a mutant (eLH/CGR-t656) of eLH/CGR, in which the C-terminal cytoplasmic tail was truncated at the Phe656 residue, through polymerase chain reaction. The eLH/CGR-t656 removed 14 potential phosphorylation sites in the intracellular C-terminal region. The plasmids were transfected into Chinese hamster ovary (CHO)-K1 and PathHunter Parental cells expressing β-arrestin, and agonist-induced cAMP responsiveness was analyzed. In CHO-K1 cells, those expressing eLH/CGR-t656 were lower than those expressing eLH/CGR wild-type (eLH/CGR-wt). The EC₅₀ of the eLH/CGR-t656 mutant was approximately 72.2% of the expression observed in eLH/CGR-wt. The maximal response in eLH/CGR-t656 also decreased to approximately 43% of that observed in eLH/CGR-wt. However, in PathHunter Parental cells, cAMP activity and maximal response of the eLH/CGR-t656 mutant were approximately 173.5% and 100.8%, respectively, of that of eLH/CGR-wt. These results provide evidence that the signal transduction of C-terminal phosphorylation in eLH/CGR plays a pivotal role in CHO-K1 cells. The cAMP level was recovered in PathHunter Parental cells expressing β-arrestin. We suggest that the signal transduction of the C-terminal region phosphorylation sites is remarkably different depending on the cells expressing β-arrestin in CHO-K1 cells.

• 한국미생물·생명공학회지
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• 제24권3호
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• pp.290-298
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• 1996

In an effort to understand the role of the conserved domain and of the heterologous one-third part of the carboxy terminal domain of transglutaminase C (TGase II), attempts were made to express TGase II cDNA of human origin in yeast Saccharomyces cerevisiae as in a full-length form as well as in a form of C-terminal truncation. The 2$\mu$-based expression plasmids which contained the TGase II cDNA under the gal inducible promoter were introduced into yeast and the maintenance of the full-length and truncated form of the TGase II gene plasmids were confirmed by Southern blot. The expression of the TGase II gene was analysed by reverse transcription polymerase chain reaction (RT-PCR), and western blot analyses. As assayed by [1,4$^{14}$C]-putrescine incorporation into succinylated casein, the full-lenth as well as the truncated forms of recombinant TGase II showed some catalytic activity. These results indicate that the N-terminal homologous domain of human TGase II retains a catalytically active domain. The level of TGase II expressed in yeast, however, was far lower than satisfactory and other expression system should be sought further chracterization of the enzyme. The negative effect of TGase II on the growth of yeast is interesting with respect to the physiological effect of TGase II in cornification of epidermal keratinocytes.