Abstract
BackgroundEscherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous.ResultsWe discovered that domain D of the Staphylococcus aureus protein A was sufficient for the secretion of various target proteins into the periplasmic space of E. coli. Our experiments indicated the Sec pathway as the mode of secretion, although N-terminal processing was not observed. Furthermore, the solubility of recombinant fusion proteins was improved for proteins prone to aggregation.The tag allowed a straightforward affinity purification of recombinant fusion protein via an IgG column, which was exemplified for the target protein human superoxide dismutase 1 (SOD).ConclusionsIn this work we present a new secretion tag that combines several advantages for the production of recombinant proteins in E. coli. Domain D of S. aureus protein A protects the protein of interest against N-terminal degradation, increases target protein solubility and enables a straight-forward purification of the recombinant protein using of IgG columns.
Highlights
Escherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities
It has been shown that the Staphylococcus aureus Protein A (SpA) secretion signal combined with miscellaneous Protein A sub-domains directs heterologous proteins into the periplasm or even to the culture supernatant [13]
We found the main portion of GFPmut3.1 either in the periplasm or in inclusion bodies
Summary
Escherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. Depending on the characteristics of the target protein, E. coli offers different compartments to meet the requirements for successful expression and purification. Cytoplasmic expression offers high yields of soluble product [5], but the purification from the cell lysate can be complex and costly. High level cytoplasmic overexpression may lead to the formation of inclusion bodies (IB). These protein aggregates simplify the purification but make in vitro refolding necessary [6,7]. In addition to promoting protein translocation to the periplasm these domains have been shown to improve folding of the target protein and to protect against N-terminal degradation [14]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
