Secretion of biologically-active human interferon-β by Bacillus subtilis

Abstract

Human interferon-β (hIFN-β) was used as a heterologous model protein to investigate the effects of the Bacillus subtilis AmyE propeptide and co-expression of PrsA in enhancing the secretion of heterologous proteins in B. subtilis. Secretion and activity of hIFN-β with AmyE propeptide increased by more than four-fold compared to that without AmyE propeptide. Moreover, under conditions of co-expressed PrsA, the secretion production and activity of hIFN-β with AmyE propeptide increased by more than 1.5-fold. AmyE propeptide and co-expression of PrsA thus have an additive effect on enhancing the production of the hIFN-β in B. subtilis.

Dimeric Structure of the Bacterial Extracellular Foldase PrsA

“…Gram-positive organisms, exported proteins commonly contain a propeptide sequence, which follows the export signal sequence and increases export efficiency (23). The AmyE propeptide from the ␣-amylase of B. subtilis is 11 amino acids long (ETANKSNELTA) and contains no proline residues.…”

“…The AmyE propeptide from the ␣-amylase of B. subtilis is 11 amino acids long (ETANKSNELTA) and contains no proline residues. When attached to other proteins, it improves their export (23,72). If PrsA is overexpressed in addition to the AmyE attachment, the protein export is further increased (23), suggesting functional interplay and possibly a direct interaction of PrsA and the AmyE propetide.…”

“…PrsA is essential under normal growth conditions in Bacillus subtilis (16) and PrsA-depleted cells are affected by decreased cell wall integrity (17,18), osmotic shock susceptibility (19), and increased sensitivity to antibiotics (20,21). Overexpression of PrsA improves the recombinant overproduction of biotechnologically important proteins (22,23). PrsA plays an important role as a folding factor of secreted proteins (14) including enzymes involved in cell wall biogenesis (18), toxins (25), and virulence factors (21,26).…”

Improvement of Heterologous Protein Secretion by Bacillus subtilis

“…The secretion production and activity of hIFN-β with propeptide increased by more than 4-fold (Fig. 4, Kakeshita et al, 2011b). The amount of secreted hIFNwww.intechopen.com β with propeptide was 3.7mg /L.…”

“…Arrowheads indicate the positions of the Pro-hIFN-β and hIFN-β. (adapted from Kakeshita et al, 2011b) In L. lactis, directed mutagenesis experiments demonstrated that the positive effect of LEISSTCDA on protein secretion was due to the insertion of negatively charged residues in the N-terminus of the mature moiety (Le Loir et al, 2001). In hIFN-α2b with AmyE propeptide, the first 10 amino acid residues of this mature protein have a net charge of -1.…”