Purification and Characterization of Novel Potent Antimicrobial Peptides, Potamin-1 and Potamin-2, from potato (Solanum tuberosum L cv. Gogu)

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항균 펩타이드들이 식물, 곤충, 양서류 그리고 포유류를 포함한 대부분 살아있는 생물의 선천적인 방어 기작에 중요한 작용을 하는 것으로 인식되고 있다. 또한 항균 펩타이드가 박테리아, 진균 그리고 심지어 바이러스에 대해서 항균/항생 작용을 가지는 것으로 알려지고 있다. 최근, 현재 상용되는 항생제에 대해서 내성을 가지는 균주의 등장으로 인하여 이를 극복하기 위한 방법으로 항균 작용을 가지는 펩타이드의 동정과 작용 기작의 연구에 대한 관심이 커지고 있다. 본 연구에서는 감자(Solanum tuberosum L cv. Gogu)에서 두 종류의 항균 펩타이드를 분리하였다. Ultrafiltration과 C_(18) RP-HPLC의 분리과정을 통해, 분자량 5600.0Da인 Potamin-1(PT-1)과 3228.5Da인 Potamin-2(PT-2)를 분리하였다. PT-1과 PT-2는 병원성 진균인 Candida albicans와 식물 잎마름병의 원인균인 Clavibacter michiganense subsp. michiganense에서 활성을 확인하였다. PT-1과 PT-2의 아미노-말단 서열을 분석한 결과, PT-1은 trypsin-chymotrypsin protease inhibitor와 homology를 가지고, PT-2는 다른 단백질이나 펩타이드들과 homology를 가지지 않는 펩타이드로 분석되었다. 그리고 PT-1과 PT-2는 70℃에서 20분간 열을 가했을 때에도 활성이 유지되었고, 게다가 사람 적혈구에 대해 독성을 일으키지 않았다. 이로써 감자에서 분리한 PT-1과 PT-2는 항균 활성을 갖는 항균 펩타이드로, 임상적인 이용을 기대할 수 있을 것이다.|Antimicrobial peptides have been recognized as playing important roles in the innate host defense mechanisms of most living organisms including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microorganism pathogens, which are resistant to currently available antibiotics, has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial peptides. Thus, this study was carried out to purify an antimicrobial peptides from the potato tubers of Solanum tuberosum L cv. Gogu. The peptides were purified by the following procedures; salt-extraction, ultrafiltration and C_(18) reverse phase chromatography. As the results, two antimicrobial peptides of were purified and confirmed Potamin-1 and Potamin-2. The molecular weight were 5600.0 Da and 3228.5 Da, respectively. PT-1 and PT-2 exerted antimicrobial activity against Candida albicans, pathogenic yeast strain and Clavibacter michganense subsp. michganense, plant late blight strain. PT-2 but not Pt-1 showed antibacterial activity against Staphylococcus aureus.
While PT-1 showed a homology with trypsin-chymotrypsin protease inhibitor, PT-2 is a novel antimicrobial peptide. The two peptides were thermostable and did not lyse human red blood cells, indicating that they may be useful as clinical agents with non-cytotoxity.
Alternative Title
감자(Solanum tuberosum L cv. Gogu)로부터 분리한 항균 펩타이드 Potamin-1과 Potamin-2의 정제와 특성
Alternative Author(s)
Kim, Jin-Young
朝鮮大學校 大學院
일반대학원 생물신소재학과
Awarded Date
Table Of Contents
초록 = 1
Ⅰ. Introduction = 5
Ⅱ. Material and Methods = 10
1. Materials = 10
2. Bacterial and fungal strains for antimicrobial activity = 10
3. Purification of antimicrobial peptide = 12
1) Extraction and isolation using ultrafiltration = 12
2) Purification of antimicrobial peptide on C_(18) HPLC = 12
4. Tricine Sodium dodecyl sulfate-polyacrylamide gel electrophoresis = 13
5. Antimicrobial assay = 13
6. Determination for protein concentration = 14
7. N-terminal amino acid sequence analysis = 15
8. Protease inhibition assays = 15
9. Chemical cross-linking = 16
10. Hemolytic activity = 16
11. Mass spectroscopy = 17
Ⅲ. Result = 18
1. Purification of Potamin-1 (PT-1) and Potamin-2 (PT-2) from potato tubers = 18
2. Characterization of PT-1 and PT-2 = 34
1) Antimicrobial activity = 34
2) Hemolytic activity of PT-1 and PT-2 = 39
3) N-terminal amino acid sequence analysis of PT-1 and PT-2 = 41
4) PT-1 as protease inhibitor = 44
5) The effect of disulfide bond on fuction of PT-1 = 44
6) Chemical cross-linking in buffer = 47
Ⅳ. Discussion = 48
Ⅴ. Reference = 52
朝鮮大學校 大學院
金珍渶. (2004). Purification and Characterization of Novel Potent Antimicrobial Peptides, Potamin-1 and Potamin-2, from potato (Solanum tuberosum L cv. Gogu).
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