Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system

Abstract

Proteins are now widely produced in diverse microbial cell factories. The Escherichia coli is still the dominant host for recombinant protein production but, as a bacterial cell, it also has its issues: the aggregation of foreign proteins into insoluble inclusion bodies is perhaps the main limiting factor of the E. coli expression system. Conversely, E. coli benefits of cost, ease of use and scale make it essential to design new approaches directed for improved recombinant protein production in this host cell. With the aid of genetic and protein engineering novel tailored-made strategies can be designed to suit user or process requirements. Gene fusion technology has been widely used for the improvement of soluble protein production and/or purification in E. coli, and for increasing peptide's immunogenicity as well. New fusion partners are constantly emerging and complementing the traditional solutions, as for instance, the Fh8 fusion tag that has been recently studied and ranked among the best solubility enhancer partners. In this review, we provide an overview of current strategies to improve recombinant protein production in E. coli, including the key factors for successful protein production, highlighting soluble protein production, and a comprehensive summary of the latest available and traditionally used gene fusion technologies. A special emphasis is given to the recently discovered Fh8 fusion system that can be used for soluble protein production, purification, and immunogenicity in E. coli. The number of existing fusion tags will probably increase in the next few years, and efforts should be taken to better understand how fusion tags act in E. coli. This knowledge will undoubtedly drive the development of new tailored-made tools for protein production in this bacterial system.

Keywords: Escherichia coli; Fh8 tag; H tag; fusion tags; protein immunogenicity; protein purification; soluble production; tag removal.

FIGURE 1

Strategies for soluble protein production in E. coli. (A) Expression vectors should be carefully selected in order to incorporate specific features that affect the protein production in E. coli such as solubility and/or affinity fusion tags, and to direct the protein synthesis to the E. coli cytoplasm or periplasm. Other important features include: the replicon, antibiotic-resistance markers, and transcriptional promoters (Jana and Deb, 2005; Sorensen and Mortensen, 2005a). (B) The optimization of expression conditions often directs the soluble protein production in E. coli, and it relies on a trial-and-error basis: to get a soluble TP, it may require the selection and testing of several engineered E. coli strains and cultivation conditions, and sometimes the initial expression vector has also to be re-designed. (C) The protein purification strategy should already be defined at the beginning when selecting the expression vector: if an affinity tag is incorporated, then a first affinity chromatography step should be conducted. On the other hand, if an affinity tag is prohibit, other strategies, namely, ion exchange, size exclusion, or hydrophobic interaction chromatography should be tested. After the first purification step, the TP may or may not be sufficiently pure. When it is not pure, further purification steps with other chromatographic strategies need to be conducted. (D–E) The protein quality is an essential requirement for many structural and functional application studies: a purified soluble protein may be aggregated, without a defined secondary structure, and it may also present a low thermal stability. Therefore, a biophysical characterization is often required before proceeding to the final protein’s application.

FIGURE 2

Schematic pathway from protein production to purification using the solubility tags and the hexahistidine (His₆) affinity tag of the comparison conducted by Costa et al. (2013a; adapted from Esposito and Chatterjee, 2006). (A) Eight tagged versions of the TP were expressed in E. coli: some fusions can end-up in the insoluble fraction whereas others remain in the soluble fraction. (B) Soluble fusion proteins are then purified by immobilized metal affinity chromatography (IMAC) using the His₆ tag and the fusion tags are removed from the TP by protease cleavage. (C) Some fusions will not cleave efficiently, resulting in a mixture of cleaved and uncleaved proteins that are difficult to separate. (D) Other fusions will cleave efficiently, and the TP remain in solution, being collected in the flow-through sample of a second IMAC purification step (as occurred with the Fh8 tag). Despite a successful protease cleavage, some TPs can become insoluble after tag removal leading to protein precipitation.

FIGURE 3

Protein purification strategy using the Fh8-HIC methodology. (A) Binding step: the Fh8-fused protein interacts with the hydrophobic matrix in the presence of calcium and at low salt concentration. This initial binding condition decreases the unspecific binding of other proteins from the E. coli extracts, which leave the column in the flow-through sample. (B) Washing step: by lowering the salts and calcium concentration, weakly interacting contaminant proteins are washed-out, and the Fh8-fused protein remains attached to the hydrophobic matrix. (C) Elution step: a calcium chelating agent, as for instance EDTA, will interfere in the calcium-dependent binding of the Fh8-fused protein, resulting in its elution from the hydrophobic matrix. The Fh8-fused protein can also be eluted by an alternative method: increasing the pH of the elution buffer to 10. This rise in the pH will promote a net charge around the fusion protein, which destabilizes the hydrophobic interactions and results in the elution of the fusion protein.

FIGURE 4

The schematic pathway from gene to antibody using the H fusion tag (Costa et al., 2013c). (A) Production of H-fused antigens in E. coli: *the antigen-codifying gene is inserted into a H-tag expression vector, and protein production and purification are optimized following the conditions presented in Figure 1. (B) E. coli endotoxins can be removed using a commercial endotoxin-removal kit or by hydrophobic interaction chromatography. (C) Purified H-fused antigens can be administrated into mice, rabbits, goats, among others, and this procedure is conducted without adjuvants. (D) The produced sera are analyzed by enzyme-linked immunosorbent assay (ELISA), Western blot, immunofluorescence assay (IFA), among others, to validate the specificity and practical application of polyclonal antibodies. Further processing may be required in order to obtain highly purified polyclonal antibodies.