Elastin-like polypeptide and γ-zein fusions significantly increase recombinant protein accumulation in soybean seeds.

Abstract

Soybean seeds are an ideal host for the production of recombinant proteins because of their high content of proteins, long-term stability of seed proteins under ambient conditions, and easy establishment of efficient purification protocols. In this study, a polypeptide fusion strategy was applied to explore the capacity of elastin-like polypeptide (ELP) and γ-zein fusions in increasing the accumulation of the recombinant protein in soybean seeds. Transgenic soybean plants were generated to express the γ-zein- or ELP-fused green fluorescent protein (GFP) under the control of the soybean seed-specific promoter of β-conglycinin alpha subunit (BCSP). Significant differences were observed in the accumulation of zein-GFP and GFP-ELP from that of the unfused GFP in transgenic soybean seeds based on the total soluble protein (TSP), despite the low-copy of T-DNA insertions and similar expression at the mRNA levels in selected transgenic lines. The average levels of zein-GFP and GFP-ELP accumulated in immature seeds of these transgenic lines were 0.99% and 0.29% TSP, respectively, compared with 0.07% TSP of the unfused GFP. In mature soybean seeds, the accumulation of zein-GFP and GFP-ELP proteins was 1.8% and 0.84% TSP, an increase of 3.91- and 1.82-fold, respectively, in comparison with that of the unfused GFP (0.46% TSP). Confocal laser scanning analysis showed that both zein-GFP and GFP-ELP were abundantly deposited in many small spherical particles of transgenic seeds, while there were fewer such florescence signals in the same cellular compartments of the unfused GFP-expressing seeds. Despite increased recombinant protein accumulation, there were no significant changes in the total protein and oil content in seeds between the transgenic and non-transformed plants, suggesting the possible presence of threshold limits of total protein accumulation in transgenic soybean seeds. Overall, our results indicate that γ-zein and ELP fusions significantly increased the accumulation of the recombinant protein, but exhibited no significant influence on the total protein and oil content in soybean seeds.