The multigene families of actinoporins (part II): Strategies for heterologous production in Escherichia coli

• We examined the genetic elements for heterologous expression of the eukaryotic genes in Escherichia coli.
• We analyzed all expression systems used for the production of actinoporins in E. coli.
• We compiled exhaustively the chromatography techniques used for recombinant actinoporins purification from E. coli.
• We examined the amino acid sequences and hemolytic activities of the actinoporins obtained from E. coli and anemones.

The sea anemone venom contains pore-forming proteins (PFP) named actinoporins, due to their purification from organisms belonging to Actiniaria order and its ability to form pores in sphingomyelin-containing membranes. Actinoporins are generally basic, monomeric and single-domain small proteins (∼20 kDa) that are classified as α-type PFP since the pore formation in membranes occur through α-helical elements. Different actinoporin isoforms have been isolated from most of the anemones species, as was analyzed in the first part of this review. Several actinoporin full-length genes have been identified from genomic-DNA libraries or messenger RNA. Since the actinoporins lack carbohydrates and disulfide bridges, their expression in bacterial systems is suitable. The actinoporins heterologous expression in Escherichia coli simplifies their production, replaces the natural source reducing the ecological damage in anemone populations, and allows the production of site-specific mutants for the study of the structure-function relationship. In this second part of the review, the strategies for heterologous production of actinoporins in Escherichia coli are analyzed, as well as the different approaches used for their purification. The activity of the recombinant proteins with respect to the wild-type is also reviewed.