Tomato cystatin SlCYS8 as a stabilizing fusion partner for human serpin expression in plants

Abstract

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.

An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves

“…benthamiana leaves. They also suggested a negative impact of protease inhibitor co-expression on IgG accumulation as observed with other proteins [30,36], strongly compensated by the heavy chain-stabilizing effect of co-secreted Sl CYS8 in upper and middle leaves.…”

“…Studies have also discussed the potential of protease inhibitors as co-expression partners to enhance the stability of clinically-useful recombinant proteins, including mammalian IgGs [13, 29–32, 36, 47, 48]. In line with these studies and with studies reporting the negative impact of host plant Cys proteases on the integrity of several recombinant proteins in plants [13, 26, 28, 31, 49], we here observed a significant stabilizing effect of tomato Sl CYS8 on the H10 antibody.…”

“…The antibody chains were flanked with an N-terminal protein secretion signal peptide derived from an embryonic mouse immunoglobulin HC-encoding gene, finally resulting in two distinct plasmids, pBI-ΩH10HC and pBI-ΩH10LC (Fig 1). Gene constructs for the protease inhibitors were assembled by Golden Gate cloning and assembled into a modified pEAQ vector [35] as described previously [36]. The coding sequences of tomato Sl CYS8 (GenBank accession no.…”

Production and characterization of in planta transiently produced polygalacturanase from Aspergillus nigerand its fusions with hydrophobin or ELP tags

“…The glycosylation process is initiated during protein translocation into the ER and the addition of N -glycans to the protein has been linked to diverse roles, including stability against denaturation and proteolysis, fine-tuning the charge and isoelectric point, regulating protein turnover [30], and has also been linked to protein activity [31]. Although some glycoproteins may accumulate well in the cytosol [32] and chloroplasts [33], and perform the required biological functions, others such as An PGI may require glycosylation for stability and will not accumulate in those compartments.…”

“…Optimization of linkers has been shown to improve both protein expression yield and biological activity [40]. A recent study comparing several linkers demonstrated the need for empirical evaluation of different linkers, and the possible beneficial effect of twistable linkers on activity of the fusion partners, possibly by allowing a reorientation of the functional domains [32]. Moreover, the addition of ELP and HFBI fusions had adverse effects on enzyme activity.…”