서지주요정보
Studies on action mechanism of tenecin 3, an insect antifungal protein, against the pathogenic fungus Candida albicans = 병원성 진균 Candida albicans에 대한 곤충 유래 항진균 단백질 tenecin 3의 작용 기작에 관한 연구
서명 / 저자 Studies on action mechanism of tenecin 3, an insect antifungal protein, against the pathogenic fungus Candida albicans = 병원성 진균 Candida albicans에 대한 곤충 유래 항진균 단백질 tenecin 3의 작용 기작에 관한 연구 / Dae-Hee Kim.
저자명 Kim, Dae-Hee ; 김대희
발행사항 [대전 : 한국과학기술원, 2001].
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등록번호

8012209

소장위치/청구기호

학술문화관(문화관) 보존서고

DCH 01001

SMS전송

도서상태

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초록정보

Tenecin 3 is a glycine-rich antifungal protein of 78 residues isolated from the hemolymph of insect Tenebrio molitor larva. Tenecin 3 was initially screened as a candidacidal protein in the hemolymph that did not inhibit the growth of Gram positive and Gram negative bacteria. The characteristic of the amino acid sequence and the antifungal specificity of tenecin 3 have raised a lot of interests for the mechanism of its antifungal action. However, the antifungal mechanism has not yet been studied due to its very low availability from natural source. To overcome this problem, bacterial expression systems were used to obtain the recombinant tenecin 3 proteins. The pET and pRSET expression vector systems provided rapid and convenient methods to generate the recombinant tenecin 3 proteins in E. coli. His-tagged fusion proteins were purified by $Ni^{2+}$-chelating affinity column chromatography. The recombinant tenecin 3 protein without any fusion was also purified by $Ni^{2+}$-chelating affinity column chromatography by taking advantage of the high histidine content of tenecin 3. These recombinant proteins all showed the same characteristics of natural tenecin 3; no antibacterial activity, yeast-type specific antifungal activity, salt-dependent antifungal activity, and non-hemolytic activity. The large quantities of the recombinant tenecin 3 proteins were easily obtained by using this expression and purification system. As an initial step in understanding the antifungal mechanism of tenecin 3, I also examined how tenecin 3 interacts with the pathogenic fungus Candida albicans to exert its antifungal action in comparison with a pore-forming mechanism, the most prevalent mechanism of antimicrobial proteins. The serial truncation experiment showed that two domains at N terminal half and C terminal half regions are sufficient for the antifungal activity. Tenecin 3 did not induce the release of a fluorescent dye trapped in the artificial membrane vesicles. It did not perturb the membrane potential of C. albicans during the initial interaction, either. These results suggest that tenecin 3 does not disrupt the lipid membrane through direct interaction such as pore-formation. Fluorescence confocal microscopy and FACScan analysis revealed that tenecin 3 is rapidly translocated into the cytoplasmic space by energy-dependent and temperature-dependent manners. This internalization is also dependent on the ionic environment and cellular metabolic (growth) state. These results suggest that internalization of tenecin 3 into the cytoplasm of C. albicans is mediated by a fungal endocytic uptake. The internalized tenecin 3 was dispersed in the cytoplasm, and the loss of the cell-viability occurred after internalization of tenecin 3. The internalized tenecin 3 induced the release of an anionic fungal vacuole specific dye, DFFDA from the vacuole, while the vacuolar membrane seems to be intact. The fact that the characteristics of tenecin 3 internalization and its effect differ from those of other known antifungal proteins suggests that tenecin 3 exerts its antifungal action through a novel mechanism.

서지기타정보

서지기타정보
청구기호 {DCH 01001
형태사항 viii, 106 p. : 부분색채삽도 ; 26 cm
언어 영어
일반주기 저자명의 한글표기 : 김대희
지도교수의 영문표기 : Young-Hoon Lee
지도교수의 한글표기 : 이영훈
학위논문 학위논문(박사) - 한국과학기술원 : 화학과,
서지주기 Reference : p. 93-98
주제 antifungal protein
tenecin 3
action mechanism
internalization
endocytosis
항진균 단백질
테네신 3
작용 기작
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