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标题：Cell-free expression tools to study co-translational folding of alpha helical membrane transporters
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作者：Nicola J. Harris ; Grant A. Pellowe ; Paula J. Booth 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2020
卷号：10
期号：1
页码：1-13
DOI：10.1038/s41598-020-66097-4
出版社：Springer Nature
摘要：Most helical membrane proteins fold co-translationally during unidirectional polypeptide elongation by the ribosome. Studies thus far, however, have largely focussed on refolding full-length proteins from artificially induced denatured states that are far removed from the natural co-translational process. Cell-free translation offers opportunities to remedy this deficit in folding studies and has previously been used for membrane proteins. We exploit this cell-free approach to develop tools to probe co-translational folding. We show that two transporters from the ubiquitous Major Facilitator Superfamily can successfully insert into a synthetic bilayer without the need for translocon insertase apparatus that is essential in vivo. We also assess the cooperativity of domain insertion, by expressing the individual transporter domains cell-free. Furthermore, we manipulate the cell-free reaction to pause and re-start protein synthesis at specific points in the protein sequence. We find that full-length protein can still be made when stalling after the first N terminal helix has inserted into the bilayer. However, stalling after the first three helices have exited the ribosome cannot be successfully recovered. These three helices cannot insert stably when ribosome-bound during co-translational folding, as they require insertion of downstream helices.