• 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.

• 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.

• 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.

• BMB Reports
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• 제32권2호
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• pp.181-188
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• 1999

4-Aminobutyrate aminotransferase plays an essential role in the 4-aminobutyric acid shunt, converting 4-aminobutyrate to succinic semialdehyde. Recombinant 4-aminobutyrate aminotransferases were overexpressed as their catalytically active forms in E. coli by coproduction with thioredoxin and their solubilities were also dramatically increased. In order to study the structural and functional aspects of the C-terminal domain of brain 4-aminobutyrate aminotransferase, we have constructed a C-terminal mutant of pig brain 4-aminobutyrate aminotransferase and analyzed the functional and structural roles of C-terminal amino acids residues on the enzyme. The deletion of five amino-acid residues from C-terminus did not interfere with the kinetic parameters and functional properties of the enzyme. Also, the deletion did not affect the dimeric structure of the protein aligned along the subunit interface at neutral pH. However, the deletion of the C-terminal region of the protein changed the stability of its dimeric structure at acidic pH. The dissociation of the enzyme acidic, facilitated by the deletion of five amino acids from C-terminus, abolished the catalytic activity.

• 한국연초학회지
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• 제23권2호
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• pp.133-140
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• 2001

PAP-I cDNA was synthesized from total RNA of Phytolacca americana leaves by RT-PCR, and then subcloned to recombinant vector pBluescript II SK-. Using PCR with primers designed in our laboratory, we could get the 9 deletion mutant PAP-I cDNA fragments. The first of the fragments was deleted by 66bp from immature N-terminal and then the rest were deleted by 90bp sequentially. Sequentially deletion mutant PAP-I cDNAs were inserted to pAc55M, on down-stream of gall promoter. Recombinant pAc55M was transformed to yeast cells, psy1 and the cells were spreaded on SC_urn-/glucose plate media. Colonies on SC_ura-/glucose plate were streaked on the same position of SC_ura-/glucose and SC_ura-/galactose plate, and we selected colonies growing on both plates, which carry non-cytotoxic deleted mutant PAP-I cDNA. We selected 4 deletion mutant PAP-I cDNAs which have not cytotoxicity.

• BMB Reports
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• 제29권5호
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• pp.411-416
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• 1996

In order to study the role of the protein shell in both iron uptake and iron core formation of ferritin, we constructed a deletion mutant of the ferritin gene and expressed the mutant gene in Escherichia coli, This mutant was obtained by introducing an amber mutation at position Pro-157 and a deletion of the 19 amino acid residues at the carboxy-terminus of the recombinant tadpole H-chain ferritin. The deleted amino acids correspond to E-helix forming the hydrophobic channel in the protein. E. coli harboring the plasmid pTHP157, which contains the deleted gene, was grown at $23^{\circ}C$ in the presence of 0.1 mM IPTG, and the induced protein appeared to be partly soluble. Nondenaturing polyacrylamide gel electrophoresis showed that the expressed mutant H-chains coassemble into holoprotein, suggesting that E-helix is not necessary for assembly of the subunits as reported for human H-chain ferritin. Its ability in iron core formation was proven in an Fe staining gel, the result disagreeing with the observation that the hydrophobic channel is necessary for iron core formation in human H-chain ferritin.

• 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.

• 한국연초학회지
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• 제23권2호
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• pp.123-132
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• 2001

In order to transform pokeweed antiviral protein cDNA to tobacco plant, total RNA was extracted from Phytolacca americana. PAP-II cDNA was synthesized from purified total RNA via RT-PCR and subcloned to recombinant vector pBluescript II SK-. 10 deletion mutant PAP-II cDNA fragments which were sequentially deleted from N-terminal by 90bp were synthesized from PAP-II cDNA except leading frame by PCR with primers designed in our laboratory. To select non-cytotoxic clone, pAc55M was constructed with yeast expression vector pAc55 and multicloning site(MCS). Sequentially deleted mutant PAP-II cDNAs were cloned on downstream of gall promoter of pAc55M. 6 non-cytotoxic deletion mutant PAP-II cDNA were selected. Selected cDNAs were cloned into plant expression vector pKGT101BH for transformation of these clones to plant through Agrobacterium tumefacience. After cloning, recombinant pKGT101BH carrying deleted mutant PAP-IIcDNA were transformed to Nicotiana tabacum cv. NC567. Transformed tobacco plants cultured on shooting and rooting media were transfered to green-house. About four weeks later, these plants were infected with physically infection using carborandum with PVY-VN strain. After 4 weeks, plants resistant to virus were selected , and seeds of these plants were gathered. Southern blot hybridization showed deleted fragments by 220bp and 420bp, so resistant ability of these plants is due to mutant PAP-II cDNA.

• BMB Reports
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• 제42권6호
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• pp.350-355
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• 2009

Although previous studies on hexokinase (HK) II indicate both the N- and C-terminal halves are catalytically active, we show in this study the N-terminal half is significantly more catalytic than the C-terminal half in addition to having a significantly higher $K_m$ for ATP and Glu. Furthermore, truncated forms of intact HK II lacking its first N-terminal 18 amino acids ($\Delta$18) and a truncated N-terminal half lacking its first 18 amino acids ($\Delta$18N) have higher catalytic activity than other mutants tested. Similar results were obtained by PET-scan analysis using $^{18}F-FDG$. Our results collectively suggest that each domain of HK II possesses enzyme activity, unlike HK I, with the N-terminal half showing higher enzyme activity than the C-terminal half.

• 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.