Introducing Selenocysteine into Recombinant Proteins in Escherichia coli

Abstract

Selenoproteins contain the 21st amino acid, selenocysteine. Selenocysteine is the only amino acid that is synthesized on its cognate tRNA, and it is inserted at specific recoded UGA stop codons via a complex translation system. Although highly similar to cysteine, selenocysteine has unique properties, including a stronger nucleophilic ability and lower reduction potential. Efforts to site-specifically incorporate selenocysteine to create recombinant selenoproteins involve a recoded UAG stop codon and expression of the necessary selenocysteine translation machinery. This article presents a protocol for expressing and purifying selenoproteins in Escherichia coli. © 2021 Wiley Periodicals LLC. Basic Protocol: Recombinant selenoprotein production in E. coli using a rewired translation system.

Keywords: protein engineering; selenocysteine; selenoprotein; synthetic biology; translational recoding.

Figure 1

Selenocysteine incorporation in bacterial cells. (A) Chemical structures of cysteine and selenocysteine, with the single difference between the structures highlighted. (B) Plasmid map of pSecUAG-Evol2 generated through SnapGene (available from the Addgene repository, cat. no. 163148). The plasmid encodes allo-tRNA^UTu2D (blue) as well as the necessary Sec machinery for incorporation using the rewired pathway. (C) In the native bacterial Sec incorporation pathway, tRNA^Sec is first serylated by SerRS and then converted to Sec-tRNA^Sec by SelA. SelB and the SECIS element bring Sec-tRNA^Sec to the ribosome to recode a UGA codon as Sec. (D) In the rewired Sec incorporation pathway, allo-tRNA^Sec follows the same aminoacylation steps as tRNA^Sec. The difference resides in elongation, when Sec-allo-tRNA^Sec is brought to the ribosome by EF-Tu to recode UAG codons as Sec.

Figure 2

Choosing an optimal position for an affinity tag. (A) Schematic of the process to strategically tag the selenoprotein for maximum yield. The number of protein subunits and the positioning of the Sec residues incorporated will affect where the affinity tag should be placed. The most optimal positioning is designated as 1, with subsequent options being less efficient. (B) Diagram describing the effect of the affinity tag position on downstream processing to achieve a clean final product.

Figure 3

Sample data. (A) Example of intact mass spectrometry data obtained using time-of-flight mass spectrometry. A single peak is observed for the protein of interest that indicates complete incorporation of Sec at the desired position. A gel insert shows that the protein was not clean, explaining the additional contaminating peak at 47,018 Da. (B) Example of the isotopic envelope observed from the mass spectrometry data of a peptide containing Sec. This differs considerably from (C) the isotopic envelope of the same peptide containing Ser instead. (Generated by P R. Baker and K. R. Clauser using http://prospector.ucsf.edu.)

Figure 4

General timeline and order of steps required to produce selenoproteins.