互补DNA
cDNA文库
生物
计算生物学
表达式(计算机科学)
分子生物学
细胞生物学
遗传学
计算机科学
程序设计语言
基因
作者
Svetlana V. Markova,Eugene S. Vysotski
出处
期刊:Methods in molecular biology
日期:2024-01-01
卷期号:: 289-306
标识
DOI:10.1007/978-1-0716-3642-8_13
摘要
The functional screening of cDNA libraries (or functional cloning) enables isolation of cDNA genes encoding novel proteins with unknown amino acid sequences. This approach is the only way to identify a protein sequence in the event of shortage of biological material for obtaining pure target protein in amounts sufficient to determine its primary structure, since sensitive functional test for a target protein is only required to successfully perform functional cloning. Commonly, bioluminescent proteins from representatives belonging to different taxa significantly differ in sequences due to independent origin of bioluminescent systems during evolution. Nonetheless, these proteins are frequently similar in functions and can use even the same substrate of bioluminescence reaction, allowing the use of the same functional test for screening. The cDNA genes encoding unknown light-emitting proteins can be identified during functional screening with high sensitivity, which is provided by modern light recording equipment making possible the detection of a very small amount of a target protein. Here, we present the protocols for isolation of full-size cDNA genes for the novel bioluminescent protein family of light-sensitive Ca2+-regulated photoproteins in the absence of any sequence information by functional screening of plasmid cDNA expression library. The protocols describe all the steps from gathering animals to isolation of individual E. coli colonies carrying full-size cDNA genes using photoprotein berovin from ctenophore Beroe abyssicola as an illustrative example.
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