Constructionand expressionof human FVII. DNA sequencing chromatogram of R147A (A) and K192A (C). After sequencing, R147A, K192A, and R147A/K192A variants demonstrated the successful alanine substitution that changed nucleotide sequences at position 147 and 192 to GCC, resulting in alanine substitution. The red underline represents GCC sequences. The green color represents nucleotide A. The blue color represents nucleotide C. The black color represents nucleotide G. The red color represents nucleotide T. Sequence alignment of R147A (B) and K192A (D) with WT FVII and primer. The codon at position R147 and K192 was similar to their primers. The star represents similar sequences. Western blot analysis of FVII variants in culture media supernatant (E). One microgram of total protein was applied to each lane. Nontransfected HEK (1 μg of total protein) was used as a negative control. Recombinant FVIIa (NovoSeven, 100 ng) was used as a positive control. The zymogen FVII and the light chain of the protease FVIIa appeared as a single band at 55 and 20 kDa, respectively. Secreted FVII protein levels in culture media supernatant (F). FVII protein levels were determined by ELISA. FVII mRNA levels of FVII variants (n = 3) (G). FVII mRNA levels were examined by qRT-PCR. Intracellular FVII proteins. After stable transfection, FVII protein levels in cell lysates were determined using ELISA (H). Data were reported as mean ± SEM (n = 3). ∗∗∗P < .001; ∗∗P < .01; ∗P < .05 in comparison between WT and each FVII variant.