Towards universal systems for recombinant gene expression

Hans Sørensen

doi:10.1186/1475-2859-9-27

Abstract

Recombinant gene expression is among the most important techniques used both in molecular and medical research and in industrial settings. Today, two recombinant expression systems are particularly well represented in the literature reporting on recombinant expression of specific genes. According to searches in the PubMed citation database, during the last 15 years 80% of all recombinant genes reported on in the literature were expressed in either the enterobacterium Escherichia coli or the methylotropic yeast Pichia pastoris. Nevertheless, some eukaryotic proteins are misfolded or inadequately posttranslationally modified in these expression systems. This situation demands identification of other recombinant expression systems that enable the proper expression of the remaining eukaryotic genes. As of now, a single universal system allowing expression of all target genes is still a distant goal. In this light, thorough experimental screening for systems that can yield satisfying quantity and quality of target protein is required. In recent years, a number of new expression systems have been described and used for protein production. Two systems, namely Drosophila melanogaster S2 insect cells and human embryonic kidney 293 (HEK293) cells stably expressing the EBNA-1 gene, show exceptional promise. The time has come to identify a few well-performing systems that will allow us to express, purify, and characterize entire eukaryotic genomes.

Highlights

Escherichia coli was the first host to be used for recombinant gene expression almost 40 years ago [1]
Based on searches of the PubMed citation database, the use of P. pastoris as an expression host has increased from 4% to 17% of the total recombinant genes reported on from 1995 to 2009 (Fig. 1)
Protein produced from a recombinant gene in E. coli as a soluble and functional product with high yield is the ideal situation for most research and industrial protein production purposes [2,3,4]

Summary

Introduction

Escherichia coli was the first host to be used for recombinant gene expression almost 40 years ago [1]. Protein produced from a recombinant gene in E. coli as a soluble and functional product with high yield is the ideal situation for most research and industrial protein production purposes [2,3,4]. Proteins derived from eukaryotes are prone to inclusion body formation and low yields This outcome can be explained by the fact that the rate of gene translation in E. coli is 4- to 10-fold higher than in eukaryotes [5]. Cultivation of P. pastoris in methanol-containing medium results in strong upregulation of the promoter of the alcohol oxidase I (AOX1) gene. This strong and tightly regulated promoter is incorporated into the majority of vectors for expression of recombinant genes in P. pastoris [9]. The N-linked glycan chains added posttranslationally to proteins produced in P. pastoris are substantially different from the modifications added by mammalian cells [10]

Findings

Discussion

Conclusions