• The Plant Pathology Journal
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• 제16권1호
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• pp.36-42
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• 2000

The coat protein gene of iris severe mosaic potyvirus, which was isolated in Korea, ISMV-K, from iris plant was cloned and its nucleotide sequence was determined. The coat protein of the virus contained 252 amino acid residues, including five potential N-glyxosylation site motifs. The coat protein of ISMV-K has 99.1% and 98.4% sequence identities with those of the Netherlands isolate of ISMV (ISMV-Ne) form crocus for the nucleotide and amino acids, respectively. The coat protein of ISMV-K has 50.4% to 60.3% nucleotide sequence identities and 47.3% to 55.7% amino acid identities with those of other 21 potyviruses, indicating ISMV to be a distinct species of the genus. The coat protein of ISMV-K was closely related with bean yellow mosaic virus and clover yellow vein virus in the phylogenetic tree analysis among the potyviruses analyzed. ISMV was easily and reliably detected from virus-infected iris leaves by RT-PCR with a set of the virus-specific primers.

• 한국식품영양과학회지
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• 제22권6호
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• pp.811-814
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• 1993

Sarcodon aspratus(Berk.) S. Ito에서 분리정제한 단백질가수분해효소의 특성을 조사하였다. 이 효소는 당을 2.1% 함유하고 있었다. Rose bengal, N-bromo succinimide, phenyl methyl sulfonyl fluoride(PMSF)와 같은 화학적 수식 시약에 효소 활성이 저해되었으며, 1차 반응속도론적 불활성 mode를 가지므로 활성 부위는 tryptophan과 serine으로 추정된다. N-말단에서 21번째 잔기까지의 아미노산 배열은 V-T-T-K-Q-T-N-A-P-W-G-L-G-N-I-S-T-T-N-K-L-으로 동정되었다.

• Journal of Microbiology and Biotechnology
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• 제15권1호
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• pp.72-79
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• 2005

A new Bacillus peptidase, bacillopeptidase DJ-2 (bpDJ-2), with molecular mass of 42 kDa and isoelectric point (pI) of 3.5- 3.7, was purified to homogeneity from Bacillus sp. DJ-2 isolated from Doen-Jang, a traditional Korean soybean fermented food. The enzyme was identified as an extracellular serine fibrinolytic protease. The optimal conditions for the reaction were pH 9.0 and $60^{\circ}C$. The first 18 amino acid residues of the N-terminal amino acid sequence of bpDJ-2 were TDGVEWNVDQIDAPKAW, which is identical to that of bacillopeptidase F (bpf). However, based on their Nterminal amino acid sequence, molecular size, and pI, it is different from that of bpf and extracellular 90 kDa. The whole (2,541 bp, full-bpDJ-2) and mature (1,956 bp, mature-bpDJ-2) genes were cloned, and its nucleotide sequence and deduced amino acid sequence were determined. The expressed proteins, full-bpDJ-2 and mature-bpDJ-2, were detected on SDSPAGE at expected sizes of 92 and 68 kDa, respectively.

• 생명과학회지
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• 제8권1호
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• pp.102-108
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• 1998

Gltcosylphosphatidylinositol (GPI)에 의해 고정된 단백질의 초기 형태는 골지체에서의 직접적인 processing을 수행하기 위한 아미노와 카르 복시 말단의 hydrophobic signal sequence를 소유하고 있다. 앞서, mouse 임파구로부터 NAD;arginine ADP-ribosyltransferase (Yac-1)가 클로닝되었으며 Yac-1 transferase의 아미노산 배열을 추정해 본 결과, hydrophobic 아미노와 카르복시 말단을 포함하고 있었으며 이는 GPI-anchroed 단백질들의 알려진 signal sequence와 일치하였다. 미 transferase는 야생형의 cDNA로 transfection된 NMU (rat mammary adenocarcinoma) cell의 표면에 존재하였으며 phosphoatidylinosotol-specific phospholipase C에 의해 방출되어졌다. 카르복시 말단의 hydrophobic sequence가 없는 돌연변이체는 수용성이며 분비성인 transferase를 생산하였다. 이러한 사실은 카르복시 말단의 sequence가 없는 돌연변이체는 수용성이며 분비성인 transferase를 생산하였다. 이러한 사실은 카르복시 말단의 sequence가 GPI의 부착에 중요함을 나타내준다.

• 한국정보과학회논문지:소프트웨어및응용
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• 제34권7호
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• pp.595-602
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• 2007

본 논문에서는 미지의 아미노산 서열이 신호 펩티다제 I에 의해 절단되는 분비성 단백질인지를 판별하고, 분비성 단백질일 경우에는 절단 위치를 예측하는 방법을 제안한다. 아미노산의 소수성을 이용한 전처리를 수행하여 분비성 단백질의 선도서열인 신호서열의 존재와 절단 위치를 추정한다. 전처리를 통해서 신호서열 아닌 서열을 초기에 제외함으로써 신호서열 예측의 정확도를 높인다. 지지벡터기계를 신호서열의 예측에 효과적으로 적용하기 위해서, 생물학적 정보와 관련된 아미노산 서열간의 거리를 제안한다. 아미노산의 세포내 위치를 예측할 수 있는 소수성 척도와 아미노산의 진화적인 관계를 나타낼 수 있는 치환행렬을 이용하여 아미노산 서열간의 거리를 정의한다. Swiss-Prot release 50 단백질 자료에 대하여 교차타당성 기법을 사용하여 실험한 결과 제안한 방법은 신호서열중에 98.9%를 신호서열로 판별하였고, 88%의 절단위치 예측정확도를 보였다. 기존의 방법과의 비교실험을 통해서 제안한 방법이 신호서열의 예측에 더욱 효과적임을 확인하였다.

• 한국식품영양학회지
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• 제8권1호
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• pp.37-42
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• 1995

The amino acid sequence of basic isozyme 55 of Horseradish Peroxidase (HRP E5) was determined by protein sequencing. HRP E5 consisted about 300 residues, and has a molecular weight of approximately 36,000 $\pm$ 500 dalton. The protein was rich In aspartic acid (14%), arginine(13%), and leucine(11%). The primary structure of HRP E5 was established by sequencing its tryptic (T1-T19) and lysylendopeptic (Al-A3) peptides. The sequence homology between HRP E5 and HRP C (neutral isozyme of horseradish peroxidase) is found to be more than 66%. The highest concentration of identical residues are found on residues 29~56, 90~123, and 155~173, but relatively low on 174~271.

• KSBB Journal
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• 제22권5호
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• pp.362-365
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• 2007

Cyanobacterial A-domain의 A3 motif와 A7 motif의 높은 진화론적 보존성에 의거해서 Non-ribosomal peitides를 생산하는 시아노박테리아를 Screening할 수 있는 degenerated primer를 만들 수 있었다. Degenerate primer서열의 종류는 가능하면 1,000개 정도까지를 기준으로 만드는 것이 좋다. Primer의 종류가 너무 많으면 primer 1종류 당 mol수가 적게 되어 특이성도 저하된다. 그러므로 Primer의 종류가 많을 경우는 inosin을 N (4종류의 염기) 부분에 이용하면 어느 염기에도 강하게 결합하지 않고 두 가닥 DNA 형성을 저해하지도 않으므로 degeneration을 줄이는데 도움이 된다. Degenerate primer의 annealing 온도는 primer에 포함되어있는 서열 중 가장 낮은 Tm을 기준으로 한다. 이번 연구처럼 N (ACGT) 대신에 Inosin을 이용하였을 때에는 Inosin이 Tm을 높게 하지 않고 Tm을 낮게 하지도 않으므로 Tm 계산시 고려하지 않아도 되었다. PCR 효율이 떨어질 우려가 있으므로 충분한 Tm값 (대개 $45\sim60^{\circ}C$ 이상)을 갖는 서열을 디자인하여 primer로 PCR하는 것이 좋지만, A3/A7 degenerate prime에서는 실험에 의해 40$^{\circ}C$로 annealing 온도가 (Tm) 다소 낮게 설정되었다. 그러므로 검출되지 않은 NRPS gene을 가진 균주와 CBT635, CBT654와 같이 약한 PCR band의 형성은 새로 제작된 primer의 낮은 Tm 기인한다고 생각되어진다. Tm의 이론적인 값은 Tm ={(G+C)*4+(A+T)*2}의 식을 통해서 정방향 primer에서 54$^{\circ}C$ 역방향 primer에서 42$^{\circ}C$로 계산되었다. 새로운 degenerate primer에 의해서 MTF2/MTR2로 검출되지 않는 6개의 균주가 더 검출되었으며, A3/A7과 MTF2/MTR2를 이용한 통합 PCR Screening을 통해서 NRPS gene 검출에 특이성과 효율성을 높일 수 있다.

• Journal of Microbiology and Biotechnology
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• 제11권1호
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• pp.29-34
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• 2001

The mutant enzymes of N-carbamyl-D-amino aicd amidohydrolase (N-carbamylase) from Agrobacterium radiobacter NRRL B11291, showing a negligible activity, were selected from the library generated by random mutagenesis. From the sequence analysis, these mutants were found to contain the amino acids substitutions at Cys172, Glu47, and Glu146. Previously, Cys172 was reported to be necessary for the enzyme catalysis. The chemical modification of the N-carbamylase by carboxyl group specific chemical reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC), resulted in a loss of activity. The replacement of glutamic acids with glutamines by site-directed mutagenesis led to aggregation of the enzymes. Mutant enzymes fused with maltose binding protein (MBP) were expressed in soluble form, but were inactive. These results indicate that two glutamic acid residues play an important role in structure and function of the N-carbamylase. Multiple sequence alignment of the related enzymes revealed that Glu47 and Glu146 are rigidly conserved, which suggests that tese residues are crucial for the structure and function of the functionally related C-N hydrolases.

• Journal of Microbiology and Biotechnology
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• 제21권2호
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• pp.129-135
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• 2011

Cloning and characterization of the gene encoding a solvent-tolerant protease from the haloalkaliphilic bacterium Geomicrobium sp. EMB2 are described. Primers designed based on the N-terminal amino acid sequence of the purified EMB2 protease helped in the amplification of a 1,505-bp open reading frame that had a coding potential of a 42.7-kDa polypeptide. The deduced EMB2 protein contained a 35.4-kDa mature protein of 311 residues, with a high proportion of acidic amino acid residues. Phylogenetic analysis placed the EMB2 gene close to a known serine protease from Bacillus clausii KSM-K16. Primary sequence analysis indicated a hydrophobic inclination of the protein; and the 3D structure modeling elucidated a relatively higher percentage of small (glycine, alanine, and valine) and borderline (serine and threonine) hydrophobic residues on its surface. The structure analysis also highlighted enrichment of acidic residues at the cost of basic residues. The study indicated that solvent and salt stabilities in Geomicrobium sp. protease may be accorded to different structural features; that is, the presence of a number of small hydrophobic amino acid residues on the surface and a higher content of acidic amino acid residues, respectively.

• 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.