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.
Highlights
Escherichia coli offers many advantages as a production organism, including growth on inexpensive carbon sources, rapid biomass accumulation, amenability to high cell-density fermentations and simple process scale-up (Mergulhao et al 2005)
Screening homologous and heterologous signal peptides for enhanced alpha hemolysin toxinH35L mutant (ATH35L) secretion in E. coli Since the selection of the signal peptide has a major impact on recombinant protein secretion in E. coli systems (Sjostrom et al 1987), three E. coli signal peptides, two from the Sec pathway and one from the signal peptide recognition particle (SRP) pathway, were screened to identify a signal peptide for efficient A TH35L secretion
A comparative analysis by Western blot showed that dsbAss was the most effective in directing the secretion of ATH35L into cell culture medium among the initial four signal peptides tested
Summary
Escherichia coli offers many advantages as a production organism, including growth on inexpensive carbon sources, rapid biomass accumulation, amenability to high cell-density fermentations and simple process scale-up (Mergulhao et al 2005). Recombinant proteins can be produced in E. coli as intracellular inclusion bodies; secretion into the extracellular environment is preferred as it simplifies downstream purification processes, protects recombinant proteins from proteolysis by cytoplasmic or periplasmic proteases, reduces endotoxin levels and contamination of the product by host proteins, In E. coli, the Sec-dependent (Sec) secretion pathway is the general secretion route and signal peptides linked to the N-terminus of recombinant proteins play a critical role in translocation and secretion. Han et al AMB Expr (2017) 7:93 peptide by signal peptidase and released into the periplasmic space where they fold into their native structure (Green and Mecsas 2016; Mergulhao et al 2005; Valent et al 1998). The SRP pathway relies on the SRP particle, which recognizes an N-terminal signal peptide with highly hydrophobic core during protein secretion and the binding affinity of the SRP particle for signal peptides increases with the hydrophobicity of the h-region of signal peptides (Green and Mecsas 2016; Nilsson et al 2015)
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