The Fh8 tag: a fusion partner for simple and cost-effective protein purification in Escherichia coli.

Downstream processing is still a major bottleneck in recombinant protein production representing most of its costs. Hence, there is a continuing demand of novel and cost-effective purification processes aiming at the recovery of pure and active target protein. In this work, a novel purification methodology is presented, using the Fh8 solubility enhancer tag as fusion handle. The binding properties of Fh8 tag to a hydrophobic matrix were first studied via hydrophobic interaction chromatography (HIC). The Fh8 tag was then evaluated as a purification handle by its fusion to green fluorescent protein and superoxide dismutase. The purification efficiency of the Fh8-HIC strategy was compared to the immobilized metal ion affinity chromatography (IMAC) using the His6 tag. Results showed that the Fh8-HIC binding mechanism is calcium-dependent in a low salt medium, making the purification process highly selective. Both target proteins were biologically active, even when fused to Fh8, and were successfully purified by HIC, achieving efficiencies identical to those of IMAC. Thus, the Fh8 acts as an effective affinity tag that, together with its previously reported solubility enhancer capability, allows for the design of inexpensive and successful recombinant protein production processes in Escherichia coli.

The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli: a comparison with the traditional gene fusion technology.

The Escherichia coli host system is an advantageous choice for simple and inexpensive recombinant protein production but it still presents bottlenecks at expressing soluble proteins from other organisms. Several efforts have been taken to overcome E. coli limitations, including the use of fusion partners that improve protein expression and solubility. New fusion technologies are emerging to complement the traditional solutions. This work evaluates two novel fusion partners, the Fh8 tag (8 kDa) and the H tag (1 kDa), as solubility enhancing tags in E. coli and their comparison to commonly used fusion partners. A broad range comparison was conducted in a small-scale screening and subsequently scaled-up. Six difficult-to-express target proteins (RVS167, SPO14, YPK1, YPK2, Frutalin and CP12) were fused to eight fusion tags (His, Trx, GST, MBP, NusA, SUMO, H and Fh8). The resulting protein expression and solubility levels were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after protein purification and after tag removal. The Fh8 partner improved protein expression and solubility as the well-known Trx, NusA or MBP fusion partners. The H partner did not function as a solubility tag. Cleaved proteins from Fh8 fusions were soluble and obtained in similar or higher amounts than proteins from the cleavage of other partners as Trx, NusA or MBP. The Fh8 fusion tag therefore acts as an effective solubility enhancer, and its low molecular weight potentially gives it an advantage over larger solubility tags by offering a more reliable assessment of the target protein solubility when expressed as a fusion protein.

A novel adjuvant-free H fusion system for the production of recombinant immunogens in Escherichia coli: Its application to a 12 kDa antigen from Cryptosporidium parvum.

The production of recombinant antigens in Escherichia coli and specific polyclonal antibodies for diagnosis and therapy is still a challenge for world-wide researchers. Several different strategies have been explored to improve both antigen and antibody production, all of them depending on a successful expression and immunogenicity of the antigen. Gene fusion technology attempted to address these challenges: fusion partners have been applied to optimize recombinant antigen production in E. coli, and to increase protein immunogenicity. Taking a 12-kDa surface adhesion antigen from Cryptosporidium parvum (CP12) by example, the novel H fusion partner was presented in this work as an attractive option for the development of recombinant immunogens and its adjuvant-free immunization. The H tag (of only 1 kDa) efficiently triggered a CP12-specific immune response, and it also improved the immunization procedure without requiring co-administration of adjuvants. Moreover, polyclonal antibodies raised against the HCP12 fusion antigen detected native antigen structures displayed on the surface of C. parvum oocysts. The H tag proved to be an advanced strategy and promising technology for the diagnosis and therapy of C. parvum infections in animals and humans, allowing a rapid and simple recombinant production of the CP12 antigen.