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

Li Tian,Samuel S M Sun,Anna Mitraki

doi:10.1371/journal.pone.0024183

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.

Highlights

Production of pharmaceutical proteins in plants has been suggested as an attractive bioreactor platform for its low cost, high yield, large-scale production and reduced health risks in comparison to traditional microbial and mammalian bioreactors, and many valuable recombinant therapeutic proteins have been expressed in transgenic plants as proof-of-concept and feasibility demonstration [1,2,3,4]
The common protein purification method used in plant bioreactors is to express target proteins in fusion with affinity tags, such as His tag and StrepII tag [6,7] for subsequent affinity purification, but it suffers from difficulty and high cost in scaling up of the required affinity chromatography
After Endo H digestion for N-linked glycans removal, the upper band disappeared and the lower band became more intense in immunoblot analysis (Figure 2B, upper panel), indicating that a portion of plant lectin (PAL) was modified by N-linked glycosylation

Summary

Introduction

Production of pharmaceutical proteins in plants has been suggested as an attractive bioreactor platform for its low cost, high yield, large-scale production and reduced health risks in comparison to traditional microbial and mammalian bioreactors, and many valuable recombinant therapeutic proteins have been expressed in transgenic plants as proof-of-concept and feasibility demonstration [1,2,3,4]. Because fusion tags may affect the bioactivity of native proteins, they are generally enzymatically removed from the final protein products by an appropriate protease. This additional cleavage step in purification results in higher cost, in addition to the potential risk of non-specific cleavage of the target protein by the added protease. High cost and difficulty in scaling-up of downstream purification of the target protein, the common involvement of affinity chromatography and protease in the purification process, has hampered its industrial scale application, a cost-effective and scale-up purification method is highly desirable for further development of plant bioreactor

Methods

Results

Conclusion