Using selenocysteine-specific reporters to screen for efficient tRNASec variants

Methods Enzymol. 2022:662:63-93. doi: 10.1016/bs.mie.2021.10.005. Epub 2021 Nov 14.

Abstract

The unique properties of selenocysteine (Sec) have generated an interest in the scientific community to site-specifically incorporate Sec into a protein of choice. Current technologies have rewired the natural Sec-specific translation factor-dependent selenoprotein biosynthesis pathway by harnessing the canonical elongation factor (EF-Tu) to simplify the requirements for Sec incorporation in Escherichia coli. This strategy is versatile and can be applied to Sec incorporation at any position in a protein of interest. However, selenoprotein production is still limited by yield and serine misincorporation. This protocol outlines a method in E. coli to design and optimize tRNA libraries which can be selected and screened for by the use of Sec-specific intein-based reporters. This provides a fast and simple way to engineer tRNAs with enhanced Sec-incorporation ability.

Keywords: Genetic code expansion; Intein-based reporter; Selenoproteins; Synthetic biology; tRNA library.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Protein Biosynthesis
  • RNA, Transfer / genetics
  • RNA, Transfer / metabolism
  • RNA, Transfer, Amino Acid-Specific* / genetics
  • RNA, Transfer, Amino Acid-Specific* / metabolism
  • Selenocysteine* / genetics
  • Selenocysteine* / metabolism
  • Selenoproteins / genetics
  • Selenoproteins / metabolism

Substances

  • RNA, Transfer, Amino Acid-Specific
  • Selenoproteins
  • tRNA, selenocysteine-
  • Selenocysteine
  • RNA, Transfer