High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives

Abstract

The ease of genetic manipulation, low cost, rapid growth and number of previous studies have made Escherichia coli one of the most widely used microorganism species for producing recombinant proteins. In this post-genomic era, challenges remain to rapidly express and purify large numbers of proteins for academic and commercial purposes in a high-throughput manner. In this review, we describe several state-of-the-art approaches that are suitable for the cloning, expression and purification, conducted in parallel, of numerous molecules, and we discuss recent progress related to soluble protein expression, mRNA folding, fusion tags, post-translational modification and production of membrane proteins. Moreover, we address the ongoing efforts to overcome various challenges faced in protein expression in E. coli, which could lead to an improvement of the current system from trial and error to a predictable and rational design.

Keywords: 5′UTR and N-terminal codons; Escherichia coli; fusion tag; high-throughput; membrane protein; recombinant protein expression.

Figure 1.

Three strategies for preparing target genes. (a) Target genes can be obtained from a cDNA library after reverse transcription. (b) PCR can be used to amplify genes from a cDNA library or genomic DNA. (c) Array-based gene synthesis through the assembly of short oligos can be used to produce customized genes.

Figure 2.

Schematic diagrams and principles of the construction of recombinant expression vectors. Target genes featuring two adapters are obtained from PCR or gene synthesis. (a) Construction of expression vectors using restriction enzymes and ligases. The vector and target genes harbouring restriction sites are digested using two rare-cutting enzymes, SgfI and PmeI. The linearized expression vector and inserts are ligated using T4 ligase to create the construct. (b) Construction of expression clones using recombination-based methods. The target genes are flanked by 15–25 bp recombination sites. Recombinase-mediated recombination between the homologous sites present in the insert and vector generates the final vector. (c) Construction of expression clones using LIC methods. Linearized vectors and target genes containing complementary 5′-tails are digested using enzymes possessing exonuclease activity in order to increase the proportion of recessed ends. The overhangs can anneal and are ligated in vivo after transformation into E. coli.

Figure 3.

Basic expression vectors for high-throughput expression in E. coli of (a) cytoplasmic proteins and (b) membrane proteins. The T7 promoter is used to control expression of the protein in E. coli. The high-throughput assay requires tandem affinity tags, larger tag for protein expression initiation, protein solubility and soluble detection, and smaller tag for purification. TEV protease can be used to remove the tags. The tags for membrane proteins are located at the C-terminus for protein targeting, and GFP is a favourable choice for use as an indicator of protein folding. D tag, detection tag; P tag, purification tag; S tag, solubility and translation initiation tag; TT, transcriptional terminator; 5′UTR, 5′ untranslated region.

Figure 4.

Escherichia coli strains for protein expression. (a) Escherichia coli strains widely used in recombinant protein production. In the expression vector, the target gene is under control of the T7 promoter. In the E. coli genome, the gene encoding T7 RNA polymerase is under control of the lacUV5. The strain BL21(DE3) is deficient in OmpT and Lon proteases. BL21STAR(DE3) is mutated in RNase E, reducing mRNA degradation. BL21trxB promotes the formation of disulfide bonds. In BL21pLysS(DE3), T7 lysozyme is expressed, and the enzyme inactivates any T7 RNA polymerase that may be produced without induction. Rosetta strains are designed to improve the expression of proteins encoded by genes containing rare codons used in E. coli. (b) Strategy for expressing a protein with post-translational modification in E. coli. Genes encoding kinases, glycosyltransferases, methylases, ligases or other modifying enzymes are coexpressed in order to produce post-translationally modified proteins. (c) Overview of E. coli strains used in membrane protein production. Walker strains (C41(DE3) and C43(DE3)) are commonly used to overcome the toxicity of membrane proteins. In Lemo21(DE3), expression can be tuned by adding different concentrations of l-rhamnose to the culture. Coexpression of membrane protein biogenesis factors may also facilitate the localization of target proteins. lysY, lysozyme; RNAP, RNA polymerase; tRS, tRNA synthetase.