REVIEW ARTICLE published: 19 February 2014 doi: 10.3389/fmicb.2014.00063 Fusion tags for protein solubility, purification, and immunogenicity in Escherichia coli: the novel Fh8 system Sofia Costa1,2 , André Almeida 3 , António Castro 2 and Lucília Domingues1* 1 Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Braga, Portugal 2 Instituto Nacional de Saúde Dr. Ricardo Jorge, Porto, Portugal 3 Hitag Biotechnology, Lad., Biocant, Parque Technologico de Cantanhede, Cantanhede, Portugal Edited by: Proteins are now widely produced in diverse microbial cell factories. The Escherichia coli Germán Leandro Rosano, Instituto de is still the dominant host for recombinant protein production but, as a bacterial cell, it Biología Molecular y Celular de Rosario, Argentina 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 Reviewed by: Grzegorz Wegrzyn, University of benefits of cost, ease of use and scale make it essential to design new approaches Gdansk, Poland directed for improved recombinant protein production in this host cell. With the aid of Helena Berglund, Karolinska genetic and protein engineering novel tailored-made strategies can be designed to suit Institutet, Sweden user or process requirements. Gene fusion technology has been widely used for the *Correspondence: improvement of soluble protein production and/or purification in E. coli, and for increasing Lucília Domingues, Institute for Biotechnology and Bioengineering, peptide’s immunogenicity as well. New fusion partners are constantly emerging and Centre of Biological Engineering, complementing the traditional solutions, as for instance, the Fh8 fusion tag that has been University of Minho, Campus de recently studied and ranked among the best solubility enhancer partners. In this review, we Gualtar, 4710-057 Braga, Portugal e-mail: [email protected] 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, fusion tags, soluble production, protein purification, tag removal, Fh8 tag, H tag, protein immunogenicity OUTLINE licensed up to 2011 by the U.S. Food and Drug Administration Proteins are key elements of life, constituting the major part of the (FDA) and European Medicines Agency (EMEA) were obtained living cell. They play important roles in a variety of cell processes, using this host cell (Ferrer-Miralles et al., 2009; Walsh,2010; Berlec including cell signaling, immune responses, cell adhesion, and the and Strukelj, 2013). cell cycle, and their failure is consequently correlated with several Escherichia coli recombinant protein-based products can also diseases. be found in major sectors of the enzyme industry and the agri- With the introduction of the DNA recombinant technology in cultural industry with applications ranging from catalysis (e.g., the 1970s, proteins started to be expressed in several host organ- washing detergents) and therapeutic use (e.g., vaccine develop- isms resulting in a faster and easier process compared to their ment) to functional analysis and structure determination (e.g., natural sources (Demain and Vaishnav, 2009). Escherichia coli crystallography; Demain and Vaishnav, 2009). remains the dominant host for producing recombinant proteins, As a bacterial system, the E. coli has, however, limitations at owing to its advantageous fast and inexpensive, and high yield expressing more complex proteins due to the lack of sophisticated protein production, together with the well-characterized genetics machinery to perform posttranslational modifications, resulting and variety of available molecular tools (Demain and Vaishnav, in poor solubility of the protein of interest that are produced as 2009). inclusion bodies (Demain and Vaishnav, 2009; Kamionka, 2011). The recombinant protein production in E. coli has greatly con- Previous studies (Bussow et al.,2005; Pacheco et al.,2012)reported tributed for several structural studies; for instance, about 90% of that up to 75% of human proteins are successfully expressed in E. the structures available in the Protein Data Bank were determined coli but only 25% are produced in an active soluble form using on proteins produced in E. coli.(Nettleship et al.,2010; Bird,2011). this host system. Other problems found within this host system The E. coli recombinant production has also boosted the biophar- include proper formation of disulfide bonds, absence of chap- maceutical industry: 30% of the recombinant biopharmaceuticals erones for the correct folding, and the miss-match between the www.frontiersin.org February 2014 | Volume 5 | Article 63 | 1 Costa et al. Fusion tags for protein production codon usage of the host cell and the protein of interest (Terpe, when inefficiently processed by molecular chaperones, promote 2006; Demain and Vaishnav, 2009; Pacheco et al., 2012). More- inclusion body formation (Sorensen and Mortensen, 2005a,b). over, the industrial culture of E. coli leads cells to grow in harsh Strategies that direct the soluble production of proteins in E. conditions, resulting in cell physiology deterioration (Chou, 2007; coli are, thus, envisaged, and become more attractive than protein Pacheco et al., 2012). refolding procedures from inclusion bodies. Despite the above-mentioned issues of E. coli recombinant pro- Several methods have been shown to prevent or decrease tein production, the benefits of cost and ease of use and scale make protein aggregation during protein production in E. coli on a it essential to design new strategies directed for recombinant sol- trial-and-error basis, including: uble protein production in this host cell. Several strategies have been made for efficient production of proteins in E. coli, namely, (i) Lower expression temperatures: bacteria cultivation at reduced the use of different mutated host strains, co-production of chaper- temperatures is often used to reduce protein aggregation, since ones and foldases, lowering cultivation temperatures, and addition it slows down the rate of protein synthesis and folding kinet- of a fusion partner (Terpe, 2006; Demain and Vaishnav, 2009). The ics, decreasing the hydrophobic interactions that are involved combination of some of these strategies has improved the soluble in protein self-aggregation (Schumann and Ferreira, 2004; production of recombinant proteins in E. coli, but the prediction Sorensen and Mortensen, 2005b). Low cultivation tempera- of robust soluble protein production processes is still a“a challenge tures can also reduce or impair protein degradation due to a and a necessity” (Jana and Deb, 2005). poor activity of heat shock proteases that are usually induced Nowadays, with the aid of genetic and protein engineering, during protein overproduction in E. coli (Chesshyre and Hip- novel tailor-made strategies can be designed to suit user or process kiss, 1989). This strategy has, however, some drawbacks requirements. as the reduction of temperature can also affect replication, This review describes the key solubility factors that correlate transcription, and translation rates, besides decreasing the with successful protein production in E. coli, and it presents a bacterial growth and protein production yields. Nevertheless, comprehensive summary of the available fusion partners for pro- these limitations can be circumvented by the use of cold- tein production and purification in the bacterial host. A main inducible promoters that maximize protein production under focus is given to the novel Fh8 fusion system (Hitag®) for soluble low temperature conditions (Mujacic et al., 1999). protein production, purification and immunogenicity in E. coli (ii) E. coli-engineered host strains: E. coli mutant strains are a (Costa, 2013). significant advance toward the soluble production of difficult recombinant proteins. Several targeted strain strategies have SOLUBLE PROTEIN PRODUCTION IN ESCHERICHIA COLI been developed through the introduction of DNA mutations The production of recombinant proteins requires a successful cor- that affect protein synthesis, degradation, secretion, or fold- relation between the gene’s expression, protein solubility, and its ing (reviewed in Makino et al., 2011), including: engineered purification (Esposito and Chatterjee, 2006). The production lev- strains for improved protein processing at low tempera- els of recombinant proteins synthesized in E. coli arenolonger tures, such as the Arctic Express strain (Agilent Technologies); pointed as a limitation for the success of the overall process, but mutated strains that increase mRNA stability by attenuation care should be taken with the protein solubility, which is still of RNases activity, which is responsible for the shorter half- a major bottleneck in the field. The