A rapid, efficient, and low-cost BiFC protocol and its application in studying in vivo interaction of seed-specific transcription factors, RISBZ and RPBF.

Proteins regulate cellular and biological processes in all living organisms. More than 80% of the proteins interact with one another to perform their respective functions; therefore, studying the protein-protein-interaction has gained attention in functional characterization studies. Bimolecular fluorescence complement (BiFC) assay is widely adopted to determine the physical interaction of two proteins in vivo. Here, we developed a simple, yet effective BiFC assay for protein-protein-interaction using transient Agrobacterium-mediated-transformation of onion epidermal cells by taking case study of Rice-P-box-Binding-Factor (RPBF) and rice-seed-specific-bZIP (RISBZ) in vivo interaction. Our result revealed that both the proteins, i.e., RISBZ and RPBF, interacted in the nucleus and cytosol. These two transcription factors are known for their coordinate/synergistic regulation of seed-protein content via concurrent binding to the promoter region of the seed storage protein (SSP) encoding genes. We further validated our results with BiFC assay in Nicotiana by agroinfiltration method, which exhibited similar results as Agrobacterium-mediated-transformation of onion epidermal cells. We also examined the subcellular localization of RISBZ and RPBF to assess the efficacy of the protocol. The subcellular localization and BiFC assay presented here is quite easy-to-follow, reliable, and reproducible, which can be completed within 2-3 days without using costly instruments and technologies that demand a high skill set.

A cost-effective ELP-intein coupling system for recombinant protein purification from plant production platform.

BACKGROUND: Plant bioreactor offers an efficient and economical system for large-scale production of recombinant proteins. However, high cost and difficulty in scaling-up of downstream purification of the target protein, particularly the common involvement of affinity chromatography and protease in the purification process, has hampered its industrial scale application, therefore a cost-effective and easily scale-up purification method is highly desirable for further development of plant bioreactor. METHODOLOGY/PRINCIPAL FINDINGS: To tackle this problem, we investigated the ELP-intein coupling system for purification of recombinant proteins expressed in transgenic plants using a plant lectin (PAL) with anti-tumor bioactivity as example target protein and rice seeds as production platform. Results showed that ELP-intein-PAL (EiP) fusion protein formed novel irregular ER-derived protein bodies in endosperm cells by retention of endogenous prolamins. The fusion protein was partially self-cleaved in vivo, but only self-cleaved PAL protein was detected in total seed protein sample and deposited in protein storage vacuoles (PSV). The in vivo uncleaved EiP protein was accumulated up to 2-4.2% of the total seed protein. The target PAL protein could be purified by the ELP-intein system efficiently without using complicated instruments and expensive chemicals, and the yield of pure PAL protein by the current method was up to 1.1 mg/g total seed protein. CONCLUSION/SIGNIFICANCE: This study successfully demonstrated the purification of an example recombinant protein from rice seeds by the ELP-intein system. The whole purification procedure can be easily scaled up for industrial production, providing the first evidence on applying the ELP-intein coupling system to achieve cost-effective purification of recombinant proteins expressed in plant bioreactors and its possible application in industry.