Production of recombinant proteins in Escherichia coli using an N-terminal tag derived from sortase

Caswell, Jill; Snoddy, Philip; McMeel, Darragh; Buick, Richard; Scott, Christopher J.

doi:10.1016/j.pep.2009.10.012

Cited by 10 publications

(4 citation statements)

References 23 publications

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“…Traditional fusion systems like MBP, GST, NusA, or Trx have constantly been challenged and complemented by novel fusion solutions such as the SUMO tag (Butt et al, 2005; Marblestone et al, 2006), the HaloTag (Ohana et al, 2009), the SNUT tag (Caswell et al, 2010), and the expressivity tag (Hansted et al, 2011), among others.…”

Section: Conclusion and Future Trendsmentioning

confidence: 99%

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

et al. 2014

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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.

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Section: Conclusion and Future Trendsmentioning

confidence: 99%

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

et al. 2014

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“…Sortase A from S. aureus is an extremely soluble enzyme that can be produced in high yield (>40 mg L −1 of culture). This property has been exploited to enhance the solubility of proteins of interest by fusion to a version of sortase lacking the catalytic cysteine 83…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Making and Breaking Peptide Bonds: Protein Engineering Using Sortase

2011

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Sortases are a class of bacterial enzymes that possess transpeptidase activity. It is their ability to site-specifically break a peptide bond and then reform a new bond with an incoming nucleophile that makes sortase an attractive tool for protein engineering. This technique has been adopted for a range of applications, from chemistry-based to cell biology and technology. In this Minireview we provide a brief overview of the biology of sortase enzymes and current applications in protein engineering. We identify areas that lend themselves to further innovation and that suggest new applications.

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“…The SrtA possesses expression-and solubilityenhancing properties (Mao 2004;Caswell et al 2010) which favor its application as a fusion tag. However, this system has a potential drawback.…”

Section: Sortase a (Srta)mentioning

confidence: 99%

Self-cleaving fusion tags for recombinant protein production

2011

Biotechnol Lett

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Fusion expression is a common practice for recombinant protein production. Some fusion tags confer solubility on the target protein whereas others provide affinity handles that facilitate purification. However, the tag usually needs to be removed from the final product, which involves using expensive proteases or hazardous chemicals and requires additional chromatography steps. Self-cleaving tags are a special group of fusion tags that possess inducible proteolytic activity. Combined with appropriate affinity tags, they enable fusion purification, cleavage and target separation to be achieved in a single step, which saves time, labor and cost. This paper reviews currently available self-cleaving fusion tags for recombinant protein production. For each system, an introduction of its key characteristics and a brief discussion of its advantages and disadvantages is given.

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Production of recombinant proteins in Escherichia coli using an N-terminal tag derived from sortase

Cited by 10 publications

References 23 publications

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

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

Making and Breaking Peptide Bonds: Protein Engineering Using Sortase

Self-cleaving fusion tags for recombinant protein production

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