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在SNAP标记活细胞中,通过化辅助的化催化铜-酸-基因循环添加

Daniel J Stone¹, Miguel Macias-Contreras¹, Shaun M Crist¹

¹Department of Chemistry and Biochemistry, Florida State University, 95 Chieftan Way, Tallahassee, FL 32306-4390, USA. lzhu@fsu.edu.

Organic & biomolecular chemistry

|September 4, 2023

概括

研究人员开发了一种新的两步方法来标记活细胞蛋白质,使用SNAP标签和化辅助铜催化亚酸循环添加 (CuAAC). 这种技术有效地用光染料标记膜蛋白,增强细胞成像能力.

科学领域:

生物化学生物化学
化学生物学化学生物学
分子生物学分子生物学

背景情况:

SNAP-tag是一种广泛用于活细胞中向蛋白质标记的酶.
现有的SNAP标签标签方法在效率和范围上可能受到限制.
铜催化亚酸环添加 (CuAAC) 是一种用于生物结合的生物对称反应.

研究的目的:

使用SNAP-tag和CuAAC.开发一种快速有效的活细胞蛋白质标记方法.
为了研究SNAP标签基质的有效性,用化酸部分进行增强标签.
为了使细胞外膜蛋白进行光标记.

主要方法:

三种SNAP标签基质的合成,包括一种具有非常规的瓜结构的基质,所有基质都含有2-picolyl azide部分.
在标准细胞培养条件下,用SNAP标签基质化活细胞.
在azide功能化的SNAP标签和乙烯基功能化的化物之间发生的铜催化亚酸循环添加 (CuAAC) 反应.
在化和非化亚基底之间标签效率的比较.

主要成果:

所有三种合成的SNAP标签基质都成功地将2-picolyl azide部分转移到活细胞中的SNAP标签.
化辅助CuAAC使膜蛋白的快速 (<1分钟) 和高效的光标记能够使用最小的铜催化剂 (20微米).
在相同的条件下,SNAP标签基板含有非化酸部分的标签效率较差.

结论:

开发的SNAP-tag/化辅助CuAAC方法快速,高效,并且与活细胞兼容.
这种方法显著扩大了SNAP-tag用于蛋白质标记的实用性,特别是对于细胞外膜蛋白.
在活细胞中使用化酸部分对于有效的SNAP标签介导的CuAAC标签至关重要.

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