Recombinant protein expression and purification

Recent advances in genomics, proteomics, and bioinformatics have facilitated the use of recombinant DNA technology in order to evaluate any protein of interest, without prior knowledge of the protein’s cellular location or function. The parallel use of affinity tags with recombinant DNA techniques, allows the facile modification of proteins of interest leading to efficient identification, production, and isolation from the host system. However, protein insolubility, conformation, stability, and structural flexibility, as well as low purification yields and host cell toxicity are challenges that must be resolved when microbial hosts are used to express recombinant proteins. To address these challenges of production and purification efficiency, fusion tags are incorporated to increase expression yields and influence solubility and native folding; novel tags in combination with affinity techniques increase purification yields; and proteases result in tag removal. In recent years, numerous fusion tags have been developed for recombinant protein production. While contemporary reports have included several overviews of available affinity tags for protein detection or purification [1–4], solubility enhancement [5], tag removal [1], or applications [6, 7] – in this comprehensive review, we provide an extensive summary of various tags and established purification strategies. We describe several design aspects for these tags that should be considered for recombinant fusion protein expression, including amino acid composition and size; Nor C-terminal fusions, used individually or in tandem; as well as optimized tags, techniques, and buffer conditions that lead to purified protein. Additionally, we discuss the availability of protocols and expression vectors through commercial vendors or DNA Review