A game of tag: A review of protein tags for the successful detection, purification and fluorescence labelling of proteins expressed in microalgae.

Recombinant proteins play a crucial role in both fundamental research and biotechnology. In the laboratory, recombinant proteins are used in a myriad of ways, including to label cells, localize proteins and isolate complexes. In the clinic, antibody-based therapeutics can dramatically increase cancer survival rates, while virus-like particles (VLPs) are being developed as next-generation vaccines. These innovations have escalated demands for biopharmaceutical recombinant proteins. However, in traditional systems (e.g. mammalian and microbial) the expression of recombinant proteins can be prohibitively expensive. One sustainable, low-cost solution is to use a microalgal-based expression system, such as Chlamydomonas reinhardtii, Phaeodactylum tricornutum, Chlorella sp., Haematococcus pluvialis or Nannochloropsis gaditana. Tools for microalgal protein expression are developing rapidly. Yet our understanding of recombinant protein expression and purification in microalgal systems lags that of traditional systems. Here, we review the impact of commonly used affinity and epitope tags (e.g. Polyhistidine-tag, Strep-tag II, HA-tag and FLAG-tag) on recombinant protein detection, purification and biofunctionality in microalgae. Additionally, we review fluorescent protein tags (such as GFP, mVenus, DsRed and mCherry) and protease cleavage sites, including 'self-cleaving' 2A peptides. Finally, we provide guidance on experimental design to enhance the likelihood of successfully expressing recombinant proteins in microalgae.