Keywords: alpha-1-antichymotrypsin, fusion proteins, peptide linkers, plant cystatins, plant-based protein expression, recombinant proteins. SummaryStudies have reported the usefulness of fusion proteins to bolster recombinant protein yields in plants. Here, we assess the potential of tomato SlCYS8, a Cys protease inhibitor of the cystatin protein superfamily, as a stabilizing fusion partner for human alpha-1-antichymotrypsin (a1ACT) targeted to the plant cell secretory pathway. Using the model expression platform Nicotiana benthamiana, we show that the cystatin imparts a strong stabilizing effect when expressed as a translational fusion with a1ACT, 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 a1ACT expressed alone. Natural and synthetic peptide linkers inserted between SlCYS8 and a1ACT have differential effects on protease inhibitory potency of the two protein partners in vitro. They also have a differential impact on the yield of a1ACT, dependent on the extent to which the hybrid protein may remain intact in the plant cell environment. The stabilizing effect of SlCYS8 does not involve Cys protease inhibition and can be partly reproduced in the cytosol, where peptide linkers are less susceptible to degradation. The effect of SlCYS8 on a1ACT 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.