Abstract
BackgroundLaboratory scale recombinant protein production and purification techniques are often complicated, involving multiple chromatography steps and specialized equipment and reagents. Here it was demonstrated that recombinant proteins can be expressed as covalently immobilized to the surface of polyester (polyhydroxyalkanoate, PHA) beads in vivo in Escherichia coli by genetically fusing them to a polyester synthase gene (phaC). The insertion of a self-cleaving module, a modified sortase A (SrtA) from Staphylococcusaureus and its five amino acid recognition sequence between the synthase and the target protein led to a simple protein production and purification method.ResultsThe generation of hybrid genes encoding tripartite PhaC-SrtA-Target fusion proteins, enabled immobilization of proteins of interest to the surface of PHA beads in vivo. After simple cell lysis and isolation of the PHA beads, the target proteins could be selectively and efficiently released form the beads by activating the sortase with CaCl2 and triglycine. Up to 6 mg/l of soluble proteins at a purity of ~98 % could be isolated in one step with no optimization. This process was used to produce and isolate three proteins: Green fluorescent protein, maltose binding protein and the Mycobacteriumtuberculosis vaccine candidate Rv1626.ConclusionsWe have developed a new technique for easy production and purification of recombinant proteins. This technique is capable of producing and purifying high yields of proteins suitable for research application in less than 2 days. No costly or specialized protein chromatography equipment, resins, reagents or expertise are required.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0385-3) contains supplementary material, which is available to authorized users.
Highlights
Laboratory scale recombinant protein production and purification techniques are often complicated, involving multiple chromatography steps and specialized equipment and reagents
Sortase transpeptidase from S. aureus can be functionally immobilized on PHA beads To first assess whether the sortase A transpeptidase for S. aureus (SrtA) could be functionally immobilized on the surface of PHA beads in vivo we made genetic fusions of the soluble form of SrtA (SrtAΔN59) [15] to the C-terminus of PhaC from Ralstonia eutropha
The activity was dependent on the presence of CaCl2 and could be removed by denaturing the protein on the beads at 95 °C for 15 min before conducting the assay, indicating that this activity is the result of the immobilized sortase
Summary
Laboratory scale recombinant protein production and purification techniques are often complicated, involving multiple chromatography steps and specialized equipment and reagents. The expression and purification of a target protein of interest is a common undertaking in many research laboratories. This task is often complicated by the multiple chromatography steps required to obtain a product of acceptable purity. Most lab-scale purification techniques require an affinity tag such as His, Strep, or GST tag to be engineered into the protein [1]. These tags require specific and often costly chromatography resins to isolate the target protein. The target protein is genetically fused to this module and bound to the affinity resin, the auto-cleaving reaction can be activated by the addition of a cofactor (e.g. metal ions) or a shift in
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.
