Expression of erythropoietin in Indian tetraploid potato variety

Priti N Desai,Harish Padh

doi:10.3410/f1000research.1-26.v1

Abstract

With the advent of protein-based biotech drugs in the market, the quest for the “perfect” protein expression system, which is both economical and effective, has come into focus. Currently bacteria, yeast, insect cells, mammalian cells, transgenic animal and transgenic plants are widely used for the expression of therapeutic proteins. Among these, transgenic plants provide advantages in terms of low production cost, lower capital investment in infrastructure, and suitable post-translational modifications. The major limitation of plants as an expression host is the low level of transgene expression. To increase the expression of heterologous proteins in plants, a number of approaches have been used. One of the approaches is to increase the transgene expression by using tissue-specific promoter(s) which can concentrate the protein of interest in targeted tissues and, thus, prove advantageous in downstream purification. In the present report, a protocol for expression of heterologous protein erythropoietin in potato tuber using patatin, the tuber-tissue-specific promoter, was standardized. Expression vectors for production of the erythropoietin gene under tissue-specific promoter were successfully constructed. For production of a transgenic plant, tissue culture techniques for regeneration of the whole plant from single explants were standardized. Polymerase chain reaction (PCR) analysis was performed to confirm the stable integration of the erythropoietin gene in the potato plant by using sequence-specific primers.

Highlights

In the past decade, transgenic plant expression systems have emerged as a serious competitive force in the large-scale production of recombinant proteins
Patatin promoter, was mobilized into Agrobacterium LBA4404 using tri-parental mating and confirmation of right clone, which was selected on the Luria agar plate containing rifampicin and chloramphenicol, and was done by the same method used for selection of the right clone in E. coli
The resultant plasmid which has the EPO gene downstream to the patatin promoter was named pPERDB33cEPO. pPERDB33cEPO was subsequently mobilized to the Agrobacterium LBA4404 using tri-parental mating and confirmed by Restriction Endonuclease (RE) digestion and agarose gel electrophoresis

Summary

Introduction

Transgenic plant expression systems have emerged as a serious competitive force in the large-scale production of recombinant proteins. The first plant-derived heterologous proteins have already reached the market[1,2] and detailed economic evaluations have demonstrated their competitiveness against established market sectors[3,4]. Several plant-derived recombinant therapeutic proteins which are in the final stage of clinical trials include human blood products, vaccine, antibody and growth hormones[5]. There have been technological developments on many levels, including transformation methods, control of gene expression, protein targeting and accumulation, the use of different crops as production platforms[6] and modifications to alter the structural and functional properties of the product. One of them is to use a tissue-specific promoter, which can concentrate the protein of interest into targeted tissue and prove advantageous during downstream purification[7]

Objectives

Methods

Results

Conclusion