An intein-based genetic selection allows the construction of a high-quality library of binary patterned de novo protein sequences.

TitleAn intein-based genetic selection allows the construction of a high-quality library of binary patterned de novo protein sequences.
Publication TypeJournal Article
Year of Publication2005
JournalProtein engineering, design & selection : PEDS
Volume18
Issue4
Pagination201-7
ISSN1741-0126
Abstract

Combinatorial libraries of synthetic DNA are increasingly being used to identify and evolve proteins with novel folds and functions. An effective strategy for maximizing the diversity of these libraries relies on the assembly of large genes from smaller fragments of synthetic DNA. To optimize library assembly and screening, it is desirable to remove from the synthetic libraries any sequences that contain unintended frameshifts or stop codons. Although genetic selection systems can be used to accomplish this task, the tendency of individual segments to yield misfolded or aggregated products can decrease the effectiveness of these selections. Furthermore, individual protein domains may misfold when removed from their native context. We report the development and characterization of an in vivo system to preselect sequences that encode uninterrupted gene segments regardless of the foldedness of the encoded polypeptide. In this system, the inserted synthetic gene segment is separated from an intein/thymidylate synthase (TS) reporter domain by a polyasparagine linker, thereby permitting the TS reporter to fold and function independently of the folding and function of the segment-encoded polypeptide. TS-deficient Escherichia coli host cells survive on selective medium only if the insert is uninterrupted and in-frame, thereby allowing selection and amplification of desired sequences. We demonstrate that this system can be used as a highly effective preselection tool for the production of large, diverse and high-quality libraries of de novo protein sequences.

URLhttps://academic.oup.com/peds/article-lookup/doi/10.1093/protein/gzi020
DOI10.1093/protein/gzi020
Short TitleProtein Eng Des Sel
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