Improved expression and secretion of protein in mammalian cells by the heterologous tissue plasminogen activator signal peptide mutation

Objective To optimize the tissue plasminogen activator (tPA) signal peptide (tPA-SP) as a universal heterologous signal peptide for the secretory expression of target proteins. Methods Two tPA-SP mutants, tPA22P/A and tPA22P/G, were constructed by substituting the amino acid proline at 22 position of tPA-SP to alanine (A) or glycine (G), respectively. Then, three HIV-1 p24 expression vectors, P24T. tPA,P24T. tPA22P/A and P24T. tPA22P/G were constructed by introducing tPA-SP, tPA22P/A or tPA22P/G into the N terminus of p24 gene. HEK293T cells were transfected by these recombinant vectors. 72h post-transfection, the p24 protein in the supernatant and cell lysate of each transfectant was examined by SDS-PAGE and western blot. Results Three p24 eukaryotic expression vectors P24T. tPA, P24T. tPA22P/A and P24T.tPA22P/G were successfully constructed. The three constructs gave secretory expression of HIV-1 p24 protein.The expression levels of p24 protein driven by P24T. tPA22P/A and P24T. tPA22P/G increased by 62% and 8% in the transferred supernatants, respectively, comparing with that driven by P24T. tPA; and the expressionlevels increased by 29% and 6% in the transferred cell lysates, respectively. Conclusions The amino acid substitution of tPA-SP 22P with alanine or glycine enhanced the expression and secretory levels of the target protein. This work provides an experimental foundation for optimizing the expression and secretion of heterologous proteins in mammalian cells. Key words: Tissue plasminogen activator;  Signal peptide;  Mammalian cells