Tomato cystatin S l CYS 8 as a stabilizing fusion partner for human serpin expression in plants

Summary Studies have reported the usefulness of fusion proteins to bolster recombinant protein yields in plants. Here, we assess the potential of tomato S l CYS 8, a C ys protease inhibitor of the cystatin protein superfamily, as a stabilizing fusion partner for human alpha‐1‐antichymotrypsin (α1 ACT ) targeted to the plant cell secretory pathway. Using the model expression platform N icotiana benthamiana , we show that the cystatin imparts a strong stabilizing effect when expressed as a translational fusion with α1 ACT , allowing impressive accumulation yields of over 2 mg/g of fresh weight tissue for the human serpin, a 25‐fold improvement on the yield of α1 ACT expressed alone. Natural and synthetic peptide linkers inserted between S l CYS 8 and α1 ACT have differential effects on protease inhibitory potency of the two protein partners in vitro . They also have a differential impact on the yield of α1 ACT , dependent on the extent to which the hybrid protein may remain intact in the plant cell environment. The stabilizing effect of S l CYS 8 does not involve C ys protease inhibition and can be partly reproduced in the cytosol, where peptide linkers are less susceptible to degradation. The effect of S l CYS 8 on α1 ACT yields could be explained by: (i) an improved translation of the human protein coding sequence; and/or (ii) an overall stabilization of its tertiary structure preventing proteolytic degradation and/or polymerization. These findings suggest the potential of plant cystatins as stabilizing fusion partners for recombinant proteins in plant systems. They also underline the need for an empirical assessment of peptide linker functions in plant cell environments.