Heterologous expression of Candida albicans Pma1p in Saccharomyces cerevisiae.
Heterologous expression of Candida albicans Pma1p in Saccharomyces cerevisiae.
复制标题
酿酒酵母中白色念珠菌PMA1P的异源表达。
DOI：
10.1111/1567-1364.12035
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发表时间：
2013-05
影响因子：
3.2
通讯作者：
Monk BC
中科院分区：
文献类型：
--
作者：
Keniya MV;Cannon RD;Nguyễn Â;Tyndall JD;Monk BC
白色念珠菌是机会性和危及生命的系统性真菌感染的主要原因，特别是在免疫功能低下的患者中。质膜质子泵ATP酶（Pma 1 p）是一种产生细胞生长所需的电化学梯度的必需酶。我们表达了C。白色念珠菌Pma 1 p（CaPma 1 p）的酿酒酵母，以促进抑制剂的筛选。取代S。酿酒酵母PMA 1与C.白色念珠菌PMA 1的克隆表达CaPma 1 p，生长缓慢，在低pH值。CaPma 1 p的表达水平显着较低，比活性低于天然Pma 1 p。它还赋予pH敏感性、潮霉素B抗性和低水平的葡萄糖依赖性质子泵。CaPMA 1和同源非必需ScPMA 2之间的相互作用导致嵌合抑制突变体表达功能性CaPma 1 p，在低pH下具有改善的H+-ATP酶活性和生长速率。抑制突变体的分子模型鉴定了可能影响S.啤酒。一种修饰的CaPma 1 p嵌合构建体，仅含有来自ScPma 2 p的5个氨基酸，能够表达用于药物筛选和结构解析的全功能酶。
Candida albicans is a major cause of opportunistic and life-threatening systemic fungal infections, especially in the immunocompromised. The plasma membrane proton pumping ATPase (Pma1p) is an essential enzyme that generates the electrochemical gradient required for cell growth. We expressed C. albicans Pma1p (CaPma1p) in Saccharomyces cerevisiae to facilitate screening for inhibitors. Replacement of S. cerevisiae PMA1 with C. albicans PMA1 gave clones expressing CaPma1p that grew slowly at low pH. CaPma1p was expressed at significantly lower levels and had lower specific activity than the native Pma1p. It also conferred pH sensitivity, hygromycin B resistance and low levels of glucose-dependent proton pumping. Recombination between CaPMA1 and the homologous non-essential ScPMA2 resulted in chimeric suppressor mutants that expressed functional CaPma1p with improved H+-ATPase activity and growth rates at low pH. Molecular models of suppressor mutants identified specific amino acids (between 531-595 in CaPma1p) that may affect regulation of the activity of Pma1p oligomers in S. cerevisiae. A modified CaPma1p chimeric construct containing only 5 amino acids from ScPma2p enabled the expression of a fully functional enzyme for drug screens and structural resolution.
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影响因子：
3.5
作者：
Lamping, E;Tanabe, K;Cannon, RD
通讯作者：
Cannon, RD
影响因子：
64.8
作者：
Auer, M;Scarborough, GA;Kühlbrandt, W
通讯作者：
Kühlbrandt, W
影响因子：
4.8
作者：
Mason, A. Brett;Allen, Kenneth E.;Slayman, Carolyn W.
通讯作者：
Slayman, Carolyn W.
DOI：
10.3109/14756360903179419
发表时间：
2010-06-01
影响因子：
5.6
作者：
Billack, Blase;Pietka-Ottlik, Magdalena;Lau-Cam, Cesar
通讯作者：
Lau-Cam, Cesar
影响因子：
3.4
作者：
Martín-Castillo, B;Portillo, F
通讯作者：
Portillo, F