Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35L in Escherichia coli

Abstract

Secretion of heterologous proteins into Escherichia coli cell culture medium offers significant advantages for downstream processing over production as inclusion bodies; including cost and time savings, and reduction of endotoxin. Signal peptides play an important role in targeting proteins for translocation across the cytoplasmic membrane to the periplasmic space and release into culture medium during the secretion process. Alpha toxinH35L (ATH35L) was selected as an antigen for vaccine development against Staphylococcus aureus infections. It was successfully secreted into culture medium of E. coli by using bacterial signal peptides linked to the N-terminus of the protein. In order to improve the level of secreted ATH35L, we designed a series of novel signal peptides by swapping individual domains of modifying dsbA and pelB signal peptides and tested them in a fed-batch fermentation process. The data showed that some of the modified signal peptides improved the secretion efficiency of ATH35L compared with E. coli signal peptides from dsbA, pelB and phoA proteins. Indeed, one of the novel signal peptides improved the yield of secreted ATH35L by 3.5-fold in a fed-batch fermentation process and at the same time maintained processing at the expected site for signal peptide cleavage. Potentially, these new novel signal peptides can be used to improve the secretion efficiency of other heterologous proteins in E. coli. Furthermore, analysis of the synthetic signal peptide amino acid sequences provides some insight into the sequence features within the signal peptide that influence secretion efficiency.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0394-1) contains supplementary material, which is available to authorized users.

Developing Gram-negative bacteria for the secretion of heterologous proteins

“…Several proteins are found primarily in the extracellular medium but do not appear to be transported by any canonical secretion pathway [32, 34, 54, 99]. These include proteins natively secreted by lab strains of E. coli such as YebF and OsmY and heterologous proteins overexpressed in E. coli such as Cel-CD from Bacillus sp.…”

“…Z-16 and α-toxin from Staphylococcus aureus . Though the full secretion mechanism is largely unknown for this class of targets, the four listed above are exported by a two-step mechanism that involves passage through the periplasm [32, 34, 54, 99] (Fig. 2b).…”

“…These fusion partners, primarily YebF and OsmY, have successfully facilitated the secretion of numerous proteins including α-amylase, leptin, IL-2, and osteopontin [100, 101]. Though titers up to 700 mg/L have been reported [102], most proteins are secreted at titers on the order of 1 mg/L or less [34, 54, 99]. Thus, engineering efforts are focused on engineering signal sequences and establishing high-throughput screening methods to allow rapid identification and engineering of factors that affect secretion titer for these fusions.…”

“…Optimizing the Sec-specific secretion signal can also increase extracellular secretion titer. Domain swapping of the common Sec signal sequences DsbA and PelB produced a chimeric signal sequence capable of increasing extracellular secretion titer of S. aureus α-toxin H35L 3.5-fold [99]. For this protein, the composition and arrangement of amino acids in the hydrophobic core of the signal sequence strongly influenced secretion titer.…”

Factors Influencing Recombinant Protein Secretion Efficiency in Gram-Positive Bacteria: Signal Peptide and Beyond

“…Substitution of Gly with Cys and Leu in the PhoE SP shifted protein secretion from SecB to SRP-dependent pathway (Adams et al, 2002). It is more likely that the order of residues and the secondary structure they formed in h-region regulate the protein secretion efficiency (Zhang et al, 2016; Han et al, 2017).…”