Figure 4.
Effect of the Sec-specific tRNASec variants and codon combinations on TrxR1 production in E. coli. By changing the natural anticodon UCA of tRNASec to either CCA or CUA, we analyzed its support of Sec insertion using expression of TrxR1 from constructs having either UAG (Stop), UGA (Stop/Sec) or UGG (Trp) at the position of Sec. Expression was performed in conjunction with a SECIS element in the construct, overexpression of the sel genes (pSUABC) or only the selC gene (pCDF-selC), and supplementation of the medium with selenite, as indicated in the figure. As for details of expression yields using the cotransformed plasmid(s), please see Table 3. The eight enzyme variants were purified to apparent homogeneity (Figures 6 and 7) whereupon turnover in three TrxR1-specific assays was determined (Trx1-coupled insulin reduction, direct DTNB reduction or PQ reduction), as indicated in the figure and further described in the text.