Figure 7.

Cell-free expression of HERV-K113 envelope variants in qRT-PCR and Western blotting analysis. K113 envelope sequences of native (K113), codon-optimized (coK113), or its mutants with rare codons (mutcoK113) were in-vitro transcribed into mRNA and then translated into protein in a cell-free expression system. Mean and standard errors were calculated from at least two (four of native and codon-optimized K113 envelope) individual experiments with GraphPad Prism5. (a) In total, 1 µg of linearized expression vector DNA was used for generation of mRNA by transcription starting at T7 promotor site upstream of ENV by RNA polymerase. A total of, 1 µg of mRNA was reverse transcribed into cDNA and used as template in qRT-PCR. The calculation of absolute mRNA levels was performed using standard curves with native or codon-optimized vector DNA, respectively. (b) In total, 4 μg of transcribed mRNA of ENV in (a) were translated into protein using reticulocyte lysate from New Zealand white rabbits including a mixture of all components necessary for translation. A translation reaction without RNA and a sample of codon-optimized RNA without amino acid methionine (L-Met) served as negative controls. An equal aliquot of all reactions was loaded for Western blotting. Densitometric analysis was performed by using ImageJ software, n=4. The de-glycosylated precursor of K113 ENV was observed at 80 kDa using anti-HERV-K-TM (HERM1811-5) antibody.