Dual-tag prokaryotic vectors for enhanced expression of full-length recombinant proteins

Abstract

The ability to express and purify proteins in large amounts through recombinant DNA technology has enabled significant advances in the biomedical sciences. Several commercially available or home-made vectors allow expression and easy purification of recombinant proteins in Escherichia coli. Generally, such vectors are designed to produce fusion proteins containing a molecular tag at the N-terminal end. Calmodulin, streptavidin, maltose binding protein, thioredoxin, hexahistidine ðHis6Þ, and glutathione S-transferase (GST) are examples of tags chosen for their high binding affinity toward specific ligands [7]. However, this procedure also has inherent limitations. In some cases, proteins overexpressed in E. coli form insoluble aggregates that are difficult to solubilize or are found mainly as truncated forms [1]. Purification of recombinant fusion proteins sometimes requires several chromatography steps and renaturation protocols. These problems are particularly evident for large proteins, for which yields of purification are frequently very low and preclude further analysis or use of the recombinant protein. Although several procedures have been developed to improve protein solubility in E. coli [1,2,6,8], less progress has been made to overcome the problem of truncated protein expression due to proteolysis, intrinsic instability, or premature translation termination caused by codon usage bias [4]. Here we describe the construction of novel dual-tag prokaryotic expression vectors that allow for enrichment in full-length recombinant proteins and facilitate their subsequent purification by sequential chromatography on metal and glutathione affinity columns. Extracellular signal-regulated kinase 3 (ERK3) is a member of the MAP kinase family of serine/threonine kinases [5]. In the course of our studies on this enzyme, we experienced problems in producing good yields of full-length GST fusion constructs of ERK3 using conventional prokaryotic expression systems. To circumvent these problems, we created a novel set of dual-tag prokaryotic expression vectors, pHGST.1 and pHGST. 2T, that were engineered to produce N-terminal His6and C-terminal GST-tagged fusion proteins (Fig. 1). In theory, the sequential purification of recombinant proteins on glutathione and metal affinity resins should: (1) allow for enrichment in full-length protein and (2) facilitate the purification process and increase the purity of the final product. pHGST vectors were constructed by first subcloning the two annealed oligonucleotides 50 CTA GCA TGA ATT CGG GAT CCA TGG GTC GAC TCG AGC TCG GAA 30 and 50 AGC TTT CCG AGC TCG AGT CGA CCC ATG GAT CCC GAA TTC ATG 30 into the NheI/HindIII sites of pRSET-A (Invitrogen Life Technologies, Burlington, Canada) to generate pRSET-MCS. The GST coding sequence was amplified by PCR using pGEX-KG [3] as template with the following oligonucleotides: 50 GCC GAG CTC TCC CCT ATA CTA GGT TAT TGG 30 and 50 CCC AAG CTT TTA ATC CGA TTT TGG AGG ATG GTC 30. The amplicon was then digested with SstI/ HindIII and subcloned into the SstI/HindIII sites of pRSET-MCS to yield pHGST.1. The pHGST.2T vector was obtained by ligation of the following annealed oligonucleotides into NcoI/XhoI-digested pHGST.1: 50 CAT GGC GGC CGC CTC GAG TCT GGT TCC Analytical Biochemistry 310 (2002) 219–222