Challenges and opportunities in the purification of recombinant tagged proteins

Abstract

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development.

Technology advancements in antibody purification

“…However, first, they must overcome the low purities that have historically been associated with these methods. 25 Thermoresponsive elastin-like polypeptide tag…”

“…They can exist as either single amino acids, short polypeptide chains, or whole proteins. 25,26 In addition to facilitating identification, affinity tags can also confer enhanced protein stability, increased expression, and facilitate native folding. 27,28 Disadvantages of affinity tags include their requirement of expensive resins as display supports, in addition to possible affinity tag removal.…”

“…32 In addition, high-molecular-weight (.100 kDa) fusions can result in partially or fully insoluble aggregates. 25,33 Epitope tags are better known for their role as antigenic determinants; however, short amino acid sequences such as poly-His (HHHHHH), C-myc (EQKLISEEDL), FLAG (DYKDDDDK), hemagglutinin (HA) (YPYDVPDYA), T7 (MASMTGGQQMG), and Glu-Glu (EYMPME) can also be used for the purification of recombinant antibodies. 28,34 These short sequences are added to the end of an antibody sequence by polymerase chain reaction-based methods.…”

“…In the presence of calcium, Einhauer and Jungbauer 47 described how an antibody (denoted here as M1) recognizes and binds FLAG-tagged recombinant antibody, which can be subsequently eluted under mild conditions, using metal chelators such as ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid. 25 In addition, they describe an anti-FLAG antibody (M2) that binds and elutes FLAGtagged antibodies in a pH-dependent manner. In 2001, Zhang et al 48 used a triple FLAG-epitope (3XFLAG tags in a row) to significantly enhance the sensitivity and detection of a mammalian expressed fusion protein using the anti-FLAG antibody M2.…”

“…Tags can be removed by two methodsfirst, by using harsh chemicals such as cyanogen bromide or hydroxylamine. 25 However, these harsh conditions may have adverse effects on the structure and/or consequently on biological activity. Secondly, a more elegant method of tag removal is the use of enzymes such as endoproteases.…”