FIGURE 2.
Analysis of recombinant proteins. A, SDS-PAGE analysis of purified recombinant proteins. After purification on a nickel-nitrilotriacetic acid column and desalting, recombinant proteins were run on a 12.5% polyacrylamide gel. 1 μg of each recombinant protein was loaded on the gel. Lanes 1–5, TGRGCUG, TRGCUG, TGRGUCG, TGRGCCG, TGRGC*, respectively; lane 6, molecular weight markers; lane 7, mtTrx; and lane 8, cTrx. The positions of molecular weight marker are indicated on the right. B and C. 75Se incorporation into recombinant TGRs. BL21(DE3) cells expressing TGRGCUG, TGRGUCG, TGRGCCG, and TGRGC* were induced with 100 μm isopropyl 1-thio-β-d-galactopyranoside for 3 h at 37 °C. 50 μCi of 75Se were added to 10-ml cultures 30 min before induction. Total cell protein samples were resolved by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. B, Coomassie Blue staining of the polyvinylidene difluoride membrane. C, 75Se detection by phosphorimaging device analysis. Lanes 1–4, 10 μl of total cell protein samples from cells expressing recombinant TGRGCUG, TGRGUCG, TGRGCCG, and TGRGC*, respectively; lane 5, molecular weight marker. FDH-O, formate dehydrogenase O (110 kDa), the single selenoprotein expressed by E. coli under aerobic conditions. The bands around 66 kDa are indicated by an asterisk on lanes 1 and 2 on the right panel and correspond to 75Se-labeled Sec-containing recombinant TGRs. Lower molecular mass bands on these lanes probably correspond to secondary initiation or degradation products of these Sec-containing recombinants. The absence of bands on lanes 3 and 4 indicates no unspecific selenium incorporation was detected. The positions of molecular weight markers are indicated on the right.