期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 434, 期 8, 页码 -出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2021.167199
关键词
genetic code expansion; tRNA; selection method; screening method; protein splicing
资金
- National Institute of General Medical Sciences [R35GM122560]
- Department of Energy Office of Basic Energy Sciences [DE-FG0298ER2031]
- Natural Sciences and Engineering Research Council of Canada (NSERC)
The presence of selenocysteine in a protein provides unique properties and makes the production of recombinant selenoproteins desirable. This study presents a versatile strategy using intein-containing reporters to screen for evolved components of a translation system yielding increased selenoprotein amounts. The results obtained with intein-containing reporters are comparable to the Sec incorporation levels determined by mass spectrometry.
The presence of selenocysteine in a protein confers many unique properties that make the production of recombinant selenoproteins desirable. Targeted incorporation of Sec into a protein of choice is possible by exploiting elongation factor Tu-dependent reassignment of UAG codons, a strategy that has been continuously improved by a variety of means. Improving selenoprotein yield by directed evolution requires selection and screening markers that are titratable, have a high dynamic range, enable high-throughput screening, and can discriminate against nonspecific UAG decoding. Current screening techniques are limited to a handful of reporters where a cysteine (Cys) or Sec residue normally affords activity. Unfortunately, these existing Cys/Sec-dependent reporters lack the dynamic range of more ubiquitous reporters or suffer from other limitations. Here we present a versatile strategy to adapt established reporters for specific Sec incorporation. Inteins are intervening polypeptides that splice themselves from the precursor protein in an autocatalytic splicing reaction. Using an intein that relies exclusively on Sec for splicing, we show that this intein cassette can be placed in-frame within selection and screening markers, affording reporter activity only upon successful intein splicing. Furthermore, because functional splicing can only occur when a catalytic Sec is present, the amount of synthesized reporter directly measures UAG-directed Sec incorporation. Importantly, we show that results obtained with intein-containing reporters are comparable to the Sec incorporation levels determined by mass spectrometry of isolated recombinant selenoproteins. This result validates the use of these intein-containing reporters to screen for evolved components of a translation system yielding increased selenoprotein amounts. (C) 2021 The Author(s). Published by Elsevier Ltd.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据