Use of Transgenic Oryzacystatin-I-Expressing Plants Enhances Recombinant Protein Production

Abstract

Plants are an effective and inexpensive host for the production of commercially interesting heterologous recombinant proteins. The Escherichia coli-derived glutathione reductase was transiently expressed as a recombinant model protein in the cytosol of tobacco plants using the technique of leaf agro-infiltration. Proteolytic cysteine protease activity progressively increased over time when glutathione reductase accumulated in leaves. Application of cysteine protease promoter-GUS fusions in transgenic tobacco identified a cysteine protease NtCP2 expressed in mature leaves and being stress responsive to be expressed as a consequence of agro-infiltration. Transgenic tobacco plants constitutively expressing the rice cysteine protease inhibitor oryzacystatin-I had significantly lower cysteine protease activity when compared to non-transgenic tobacco plants. Lower cysteine protease activity in transgenic plants was directly related to higher glutathione reductase activity and also higher glutathione reductase amounts in transgenic plants. Overall, our work has demonstrated as a novel aspect that transgenic tobacco plants constitutively expressing an exogenous cysteine protease inhibitor have the potential for producing more recombinant protein very likely due to reduced activity of endogenous cysteine protease activity.

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

“…One approach along this line consists of silencing host protease forms using DNA antisense or RNAi sequences [26–28]. Another approach consists of co-expressing accessory protease inhibitors with the protein of interest to inhibit endogenous protease activities in situ [29, 30]. Co-secretion of tomato cystatins Sl CYS8 or Sl CYS9, two Cys protease inhibitors, was shown for instance to improve the accumulation of a transiently expressed diagnostic monoclonal IgG in N .…”

Proteolysis of recombinant proteins in bioengineered plant cells

“…Sl CYS9, an inhibitor of papain- and legumain-like cysteine proteases, had no impact on apoplast-based production, but stabilized the C5–1 antibody in planta, presumably upstream in the secretory pathway 19 . In our group, we investigated the use of an “in-built” protein stabilizing agent in genetically engineered tobacco plants expressing OC-I in the cytosol 52 . Constitutively expressing the rice cystatin in tobacco leaves lowered overall cysteine protease activity and increased the amounts produced of enzymatically active recombinant glutathione reductase, which was used as a model enzyme, when the enzyme was transiently produced in transgenic tobacco leaves after agroinfiltration.…”

“…Better knowledge of protease involvement in senescence processes might be particularly helpful to improve protein stability in any transient expression system, as almost all protease families have been associated with some aspects of plant senescence 56 . There is evidence that leaf infiltration with Agrobacterium cells causes leaf senescence resulting in the expression of senescence-related proteases including cysteine proteases 52 . Senescence, the final developmental stage of every plant organ, leads to cell death and senescence-associated proteolysis naturally enables the remobilization of nutrients but might also degrade recombinant proteins…”

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

“…An additional factor determining final yields is the overall stability of the recombinant polypeptide in a given cellular context. Proteolytic degradation is often cited as a yield-limiting problem in plant-based expression systems (Benchabane et al, 2008), and a number of studies have shown that co-expression of protease inhibitors can improve protein yields by reducing the impact of endogenous proteolysis (Goulet et al, 2012;Komarnytsky et al, 2006;Pillay et al, 2012;Rivard et al, 2006). While plant cells are able to process, fold and assemble complex mammalian proteins, it is also likely that these processes have a considerable influence over expression levels.…”