• Journal of Plant Biology
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• 제36권4호
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• pp.391-398
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• 1993

식물에서도 동물에서와 같이 RGD(Arg-Gly-Asp) sequence를 매개로 원형질막과 extracellular matrix(ECM)가 연결되어 있다는 사실이 알려지고 있으므로 RGD sequence가 식물세포의 생장, 분화 등의 여러 가지 생리적인 현상에 관여하리라는 가능성을 생각할 수 있다. 그래서 RGD sequence가 당근(Daucus carota L.) 배양세포에서 에틸렌 생성에 미치는 영향을 조사하였다. RGD sequence를 포함하는 합성 peptide는 당근 배양세포에서 에틸렌의 생성을 촉진시켰다. RGD peptide는 Indole-3-acetic acid(IAA)와 함께 처리한 경우 에틸렌 생성을 더욱 촉진시켰으며, ACC synthase의 활성도 증가시켰다. RGD sequence가 에틸렌 생성을 촉진시킬 때 어떠한 특이성이 있는가를 확인하기 위해 이와 유사한 RGE(Arg-Gly-Glu) peptide, RGK(Arg-Gly-Lys) peptide를 각각 처리하여 보았다. RGE peptide는 RGD peptide와 마찬가지로 에틸렌 생성을 촉진시켰으나 RGK peptide는 에틸렌 생성에 영향을 미치지 않았다. 따라서 RGD peptide의 에틸렌 생성 촉진작용은 RGD dequence를 포함하는 RGD peptide가 ACC synthase의 활성을 증가시켜서 일어나는 현상이라 생각되며, 또한 특정한 아미노산 서열을 요구하는 특이적 반응이라고 생각된다.

• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.399-405
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• 1998

From the sequence alignment of various non-ribosomal peptide synthetases, several motifs of highly conserved sequences have been identified within each domain of peptide synthetases. We designed PCR primers based on the highly conserved nucleotide sequences to amplify and isolate a ∼7.2-kb DNA fragment of the Bacillus subtilis 713 which was isolated and reported to produce an antifungal peptide compound. Nucleotide sequence analysis of 4.8 kb of the predicted amino acids revealed significant homology to various peptide synthetases over the whole sequence and also revealed two amino acid-activating domains with highly conserved Core 1 to Core 6 and spacer motif. This suggests that the isolated DNA fragment is part of a peptide synthetase gene for antifungal peptide.

• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.310-317
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• 1998

Peptide synthetases are large multifunctional enzyme complexes that catalyze the nonribosomal synthesis of a structurally diverse family of peptide antibiotics. These enzymes are composed of functionally independent domains with independent enzymatic activities. Their specific linkage order of domains forms the protein template that defines the sequence of the incorporated amino acids. Within each domain, several motifs of highly conserved sequences have been identified from the sequence alignment of the various peptide synthetases ［30］. Taking advantage of the conserved nucleotide sequence of Core 1 and Core 2, we designed PCR primers to amplify the peptide synthetase genes from three different gram-positive bacterial strains. Nucleotide sequence analysis of the amplified PCR products from those three strains showed significant homology to various peptide synthetase genes, suggesting that the PCR products are parts of peptide synthetase genes. Therefore, this rapid and efficient PCR technique can be used for the isolation of peptide synthetase genes from various strains.

• BMB Reports
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• 제37권4호
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• pp.460-465
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• 2004

A 16-residue polypeptide model with the sequence acetyl-YALSLAATLLKEAASL-OH was derived by rational de novo peptide design. The designed sequence consists of amino acid residues with high propensity to adopt an alpha helical conformation, and sequential order was arranged to produce an amphipathic surface. The designed sequence was chemically synthesized using a solid-phase method and the polypeptide was purified by reverse-phase liquid chromatography. Molecular mass analysis by electro-spray ionization mass spectroscopy confirmed the correct designed sequence. Structural characterization by circular dichroism spectroscopy demonstrated that the peptide adopts the expected alpha helical conformation in 50% acetonitrile solution. Liposome binding assay using Small Unilamellar Vesicle (SUV) showed a marked release of entrapped glucose by interaction between the lipid membrane and the tested peptide. The channel-forming activity of the peptide was revealed by a planar lipid bilayer experiment. An analysis of the conducting current at various applied potentials suggested that the peptide forms a cationic ion channel with an intrinsic conductance of 188 pS. These results demonstrate that a simple rational de novo design can be successfully employed to create short peptides with desired structures and functions.

• Animal cells and systems
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• 제5권3호
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• pp.199-203
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• 2001

Sperminogen was purified from the acid extracts of boar spermatozoa and partial peptide sequence of the 34-36 kd sperminogen was determined. Acid extracts of boar spermatozoa was gel-filtered through Sephadex G-75, and the 34-36 kd sperminogen was purified by preparative SDS-PAGE. The sperminogen bands were sliced out, and 34-36 kd sperminogen were eluted from the gel fragments and was subjected to peptide sequencing. Since the amino termini were blocked for Edman degradation method, internal amino acid sequences of the eluted 34-36 kd sperminogen were obtained from CNBr-digested peptides of sperminogen. Among several bands resolved on tricine SDS-PAGE, 14, 22 and 26 kd peptides were subjected to peptide sequencing. The ana1yzed amino acid sequences of the 26 and 22 kd peptides showed high homologies with that of the zona pellucida binding protein, Sp38, and the analyzed amino acid sequence of the 14 kd peptide showed neither sequence homology nor similarity with any known proteins.

• The Plant Pathology Journal
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• 제15권6호
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• pp.313-318
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• 1999

Cloning of the 5'untranslated region (5' UTR) and Nterminus of the glycoprotein precursor (G2G1) open reading frame of tomato spotted wilt virus has been problematic, possibly because of the toxicity of a signal peptide at the beginning of th G2G1 protein precursor. The toxicity of the signal peptide to bacterial growth and the reason for the expression of the peptide gene in Escherichia coli were investigated by cloning the 5' UTR and the signal peptide sequence separately. Cells transformed with the plasmid containing both the first 30 amino acids of the glycoprotein and the 5' UTR showed a severe growth inhibition whereas transformants harboring either the plasmid with the signal sequence or the 5'UTR alone did not show any ingibition. An E. coli promoter-like sequence was found in the 5'UTR and tis promoter acivity was confirmed with a promoter-less GUS gene cloned downstream of the 5'UTR. In the cloning of the Tomato spotted wilt virus (TSWV) glycoprotein G2G1 open reading frame all the recovered plasmids contained stop codons in the signal sequence region. However, clones containing no stop codon were recovered when the signal sequence and the 5'UTR were cloned separately.

• Journal of Microbiology and Biotechnology
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• 제9권3호
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• pp.276-281
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• 1999

In order to determine the functional region of the antifungal protein (AFP) isolated from Aspergillus giganteus responsible for growth inhibitory activity and the promotion of phospholipid vesicle aggregation, overlapping peptides covering the complete sequence of AFP were synthesized. The antibiotic activity against bacterial, fungal, and tumor cells, and the vesicle-aggregation activity of the synthetic peptides were investigated. The AFP functional sequence responsible for antibiotic and vesicle-aggregation activity was determined to be located within the region between AFP residues 19 to 32. AFP (19-32) exhibited an a-helical conformation in a cell membrane-like environment. AFP (19-32) displayed potent antibiotic activity against bacterial, fungal, and tumor cells without peptide toxicity as indicated by hemolysis. Accordingly, AFP (19-32) could be used as a good model for the design of effective antibiotic agents with powerful antibiotic activity yet without any cytotoxic effects against the host organism.

• Macromolecular Research
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• 제17권8호
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• pp.597-602
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• 2009

Although the existing design principle of full-sequence ionic complement is convenient for the development of peptides, it greatly constrains the exploration of peptides with other possible assembly mechanisms and different yet essential functions. Herein, a novel designed half-sequence ionic complementary peptide (referred to as P9), AC-Pro-Ser-Phe-Asn-Phe-Lys-Phe-Glu-Pro-$NH_2$, is reported. When transferred from pure water to sodium chloride solution, P9 underwent a dramatic morphological transformation from globular aggregations to nanofibers. Moreover, the rheological experiment showed that the P9 could form a hydrogel with a storage modulus of about 30 Pa even at very low peptide concentration (0.5% (wt/vol)). The P9 hydrogel formed in salt solution could recover in a period of about 1,800 sec, which is faster than that in the pure water. The data suggestcd that the half-sequence, ionic complementary peptide might be worthy of further research for its special properties.

• 한국수산과학회지
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• 제34권2호
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• pp.164-172
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• 2001

본 연구는 김의 Maxazyme NNP 가수분해물로부터 여러 단계의 column chromatography 및 HPLC를 통해 ACE 저해 peptide를 분리, 정제하여 이의 분자량과 amino acid sequence를 분석하였다. ACE 저해 효과가 큰 저분자 peptide의 함량이 많은 가수분해물을 얻기 위한 최적의 단백질 가수분해 효소를 선정하기 위하여 시험한 결과, Maxazyme NNP에 의한 가수분해물의 ACE 저해효과가 $37.2\%$로 가장 높게 나타났다 김의 효소 가수분해물로부터ACE 저해 peptide만을 효율적으로 분리하기 위한 추출 조건 시험에서, 색소 제거를 위해서는 diethylether 처리가, 다당류 및 고분자 단백질 제거를 위해서는 $70\%$ ethanol 처리가 각각 선정되었다. 김 가수분해물로부터 ACE 저해 peptide를 분리, 정제하기 위한 첫 단계로 ultrafiltration을 한 결과, 분자량 3,000 이하 분획물의 ACE 저해 효과가 $69.4\%$로 가장 높았으며, 이 분획물을 gel filtration chromatography (Sephadex G-25) , reverse-phase HPLC (ODS & Vydac C-18) 및 gel permeation chromatography (Superdex Peptide HR)와 같은 단계별 column chromatography를 행하여 최종적으로 단일 peptide peak들을 분리하였다. 이들 단일 peptide peak들의 분자량을 Electrospray-Mass Spectrometer로 측정한 결과, 각각 413.48 (S1O2V2V1P),346.86 (S1O2V2V2P) 그리고 320.32 (S2O6V3V1P) dalton이었으며, 이들 peptide의 amino acid sequence는 N-말단으로부터 아미노산 잔기를 분석한 결과, 각각 Val-Gln-Gly-Asn, Thr-Glu-Thr 및 Phe-Arg으로 확인되었다.

• Journal of Animal Science and Technology
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• 제50권2호
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• pp.177-184
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• 2008

Acid-labile subunit(ALS)는 85-kDa 크기의 당단백질로서 7.5-kDa의 insulin-like growth factor(IGF) 및 40~45-kDa IGF-binding protein-3와 결합하여 150-kDa ternary complex를 형성하는 혈장단백질이다. 선행연구에서 본 연구진은 reverse transcription-polymerase chain reaction(RT-PCR) 방법으로 돼지(porcine) ALS(pALS)의 coding sequence를 합성하여 plasmid vector에 삽입시켜 ‘expression construct’를 제작한 바 있다. 그러나 본 expression construct의 pALS coding sequence에는 PCR error로 추정되는 원인으로 말미암아 2개의 bases에서 mis-sense mutation이 일어난 것이 발견되었다. 본 연구에서는 ‘site-directed mutagenesis’ 방법으로 pALS의 올바른 coding sequence를 합성하여 ‘insert DNA’의 마지막 codon 다음에 ‘His-tag’ sequence가 위치한 pET- 28a(+) plasmid expression vector에 삽입하였다. 본 expression construct는 E. coli BL21(DE3) 세포에서 ‘induction’ 시켰고, 발현된 유전자재조합(recombinant) peptide는 Ni-affinity chromato- graphy로 정제하였다. 이렇게 affinity chro- matography로 정제된 peptide는 SDS-PAGE에서 66kDa 위치에 single band를 나타냄으로써 recombinant pALS의 예상된 질량과 일치하였다. 이상의 결과는 본 연구에서 recombinant pALS peptide가 성공적으로 발현정제되었음을 시사한다.