A Cost-Effective ELP-Intein Coupling System for Recombinant Protein Purification from Plant Production Platform

Abstract

BackgroundPlant 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 FindingsTo 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/SignificanceThis 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.

Production of Pharmaceutical Proteins in Solanaceae Food Crops

“…An increase in research into purification strategies has given rise to various techniques for purifying specific proteins derived from particular host production platforms. For example, Azzoni et al [108] reported recovery of 49% of aprotinin (a protease inhibitor) extracted from transgenic maize using a trypsin-agarose column and purification of recombinant proteins from seeds using oleosin partitioning [109] or ELP-Intein Coupling System in rice grains [110] were also described.…”

Molecular farming on rescue of pharma industry for next generations

“…The recombinant protein is then recovered by a series of selective and reversible processes known as Inverse Transition Cycling that includes sequential steps of precipitation, salting, heating, centrifugation and resolubilization (Meyer & Chilkoti, 1999). Recent findings highlight the potential of ELP-intein coupling system as a cost-effective purification approach at the industrial level (Tian & Sun, 2011). To improve ELP-based purification methods, some modifications have been suggested.…”

Inteins—A Focus on the Biotechnological Applications of Splicing-Promoting Proteins

“…moricin CM4, human β-defensins, which are able to disrupt bacterial cells but have no negative effects on normal mammalian cells [68]), as well as the purification of a plant lectin (PAL) that has been shown to possess anti-tumor activity [69]. Despite relatively low yields of target protein produced in currently available systems, the methodology has strong potential to become a powerful technique for heterologous protein production especially in E. coli [68] [69]. Apart from low yields, current limitations of this approach are the control of the splicing reaction (i.e.…”

“…Apart from low yields, current limitations of this approach are the control of the splicing reaction (i.e. the splicing reaction may occur too early during the expression thus lowering the final yield of purified product), as well as the temperatures required to induce ELP-mediated aggregation [58] [68] [69]. Since the stability of different target proteins may vary considerably the methodology needs to be optimized case by case.…”