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.

Characterization of the termini of the West Nile virus genome and their interactions with the small isoform of the 2′ 5′-oligoadenylate synthetase family

“…Although the mechanism of solubility enhancement by GB1 and NusA, is unclear it is hypothesized that they may act as chaperones, promoting folding and preventing aggregation of partially folded proteins, or they form micelle--like structure thereby sequestering misfolded and unfolded proteins from solvent interactions and allowing an outward presentation of soluble domain face [342]. Streptavidin and 8XHis tags allow affinity purification from cell lysate through use of either a biotin or nickel--affinity column.…”

Expression and purification of human IL-2 protein from Escherichia coli

“…Incorporation of fusion tags was reported to increase the solubility of the expressed protein and subsequently could prevent formation of IBs (Costa et al, 2014;Sorensen and Mortensen, 2005b). Therefore, a dual Histagged IL-2 protein (His-IL-2-His) was expressed to decrease the chance of IBs formation.…”

A combinatorial approach of N-terminus blocking and codon optimization strategies to enhance the soluble expression of recombinant hIL-7 in E. coli fed-batch culture

“…Strategies like lowering the cultivation temperature (Vera et al 2007), co-expression of chaperones, use of weak or moderate promoters, or application of signal sequences for protein export were partially successful to improve protein solubility (Sletta et al 2007). The Nterminal fusion of solubility enhancer tags such as thioredoxin (Trx), maltose-binding protein (MBP), small ubiquitin-like modifier (SUMO), and N-utilization substance (NusA) enhanced soluble expression of many recombinant proteins (Costa et al 2014). Various therapeutically important proteins have been expressed in a soluble form using the SUMO fusion tag like human fibroblast growth factor-21 (FGF-21) (Wang et al 2010), IFN-α (Peciak et al 2014), and hIFN-γ (Tileva et al 2016).…”