Sequence-based prediction of permissive stretches for internal protein tagging and knockdown
Internal tagging of proteins by inserting small functional peptides into surface accessible permissive sites has proven to be an indispensable tool for basic and applied science. Permissive sites are typically identified by transposon mutagenesis on a case-by-case basis, limiting scalability and their exploitation as a system-wide protein engineering tool.
We developed an apporach for predicting permissive stretches (PSs) in proteins based on the identification of length-variable regions (regions containing indels) in homologous proteins.
We verify that a protein's primary structure information alone is sufficient to identify PSs. Identified PSs are predicted to be predominantly surface accessible; hence, the position of inserted peptides is likely suitable for diverse applications. We demonstrate the viability of this approach by inserting a Tobacco etch virus protease recognition site (TEV-tag) into several PSs in a wide range of proteins, from small monomeric enzymes (adenylate kinase) to large multi-subunit molecular machines (ATP synthase) and verify their functionality after insertion. We apply this method to engineer conditional protein knockdowns directly in the Escherichia coli chromosome and generate a cell-free platform with enhanced nucleotide stability.
Functional internally tagged proteins can be rationally designed and directly chromosomally implemented. Critical for the successful design of protein knockdowns was the incorporation of surface accessibility and secondary structure predictions, as well as the design of an improved TEV-tag that enables efficient hydrolysis when inserted into the middle of a protein. This versatile and portable approach can likely be adapted for other applications, and broadly adopted. We provide guidelines for the design of internally tagged proteins in order to empower scientists with little or no protein engineering expertise to internally tag their target proteins.
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- BMC Biology
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- November 16, 2017
Keywords: Permissive site, Internal protein tagging, TEV protease, Protein knockdowns, Cell-free biotechnology