In vivo polyester immobilized sortase for tagless protein purification

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.

AL-PHA beads: bioplastic-based protease biosensors for global health applications

“…2D; Supplementary Fig. 5) also enable us to estimate that this equates to ~37 pmoles of GFP being released from ~302,000 AL-PHA beads, which is within the range of previous granule surface protein coverage estimates [65,68]. Whilst, as expected, the supernatant fluorescence levels of control biosensor samples were unchanged by AcTEV (10…”

“…Interestingly, these PHAs beads can also be functionalised in vivo with engineered fusion proteins, which decorate the materials surfaces [64]. Such functionalised PHAs beads have enabled novel protein purification strategies [65], as well as the development of PHAs-based vaccines [66]. Indeed, insights from previous studies have demonstrated that post purification, PhaC remains bound to the bead surface [67].…”

Polyhydroxyalkanoate‐associated phasins as phylogenetically heterogeneous, multipurpose proteins

“…The properties and functionalities of the PHAs depend on their monomer composition: whereas scl‐PHAs show thermoplastic properties similar to polypropylene, mcl‐PHAs display elastic features similar to rubber or elastomer (Keshavarz and Roy, ; Park et al ., ). Applications of PHAs in the industry are widespread, ranging from the manufacturing of packages and covers to the generation of enantiomeric pure chemicals (Philip et al ., ) or as protein immobilization supports (Draper and Rehm, ; Dinjaski and Prieto, ; Hay et al ., ). Of significant relevance is the implementation of PHAs in the biomedical discipline, especially supported by the recent FDA approval for P(4HB) to be used as suture material (Tepha Inc., MA, USA).…”

Recombinant surface engineering to enhance and expand the potential of biologically produced nanoparticles: A review

“…Moreover, streptavidin displayed on the granule surface was demonstrated to participate in the biotinylation of proteins and in the purification of biotinylated DNA [57]. A modified sortase A (self-cleaving module) and its five-amino acid recognition sequence, too, were fused to synthase and displayed on granules [58]. Such engineered granules have provided a simple protein production and purification method.…”