Refinement of the Fusion Tag PagP For Efficient Inclusion Body-Targeted Expression of Recombinant Antimicrobial Peptides in Escherichia Coli
Abstract The methods developed for efficient insoluble protein production are less well explored. Our data demonstrated that PagP, an E. coli outer membrane protein with high β-sheet content, could function as an efficient fusion partner for inclusion body-targeted expression of antimicrobial peptide Magainin II, Metchnikowin and Andropin. The primary structure of a given polypeptide determines to a large extent its propensity to aggregate. The aggregation “hot spots” (HSs) in PagP was subsequently analyzed with the web-based software AGGRESCAN, leading to identification of the C-terminal region with high dense distribution of HSs. The absolute yields of recombinant antimicrobial peptide Metchnikowin and Andropin could be increased significantly when expressed in fusion with this version of PagP. Moreover, a Proline-rich region was found in the β-strands of PagP. Substitution for these prolines by residues with high β-sheet propensity and hydrophobicity significantly improved its ability to form aggregates, and greatly increased the yield of the recombinant passenger peptides. Fewer examples have been presented to separate the recombinant target proteins expressed in fusion inclusion bodies. Here, we reported an artificial linker peptide NHT with three motifs, by which separation and purification of the authentic recombinant antimicrobial peptides could be implemented.
