Integrated recombinant protein expression and purification platform based on Ralstonia eutropha.
Protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. We describe a protein purification scheme wherein Ralstonia eutropha is used to produce its own "affinity matrix," thereby eliminating the need for external chromatographic purification steps. This approach is based on the specific interaction of phasin proteins with granules of the intracellular polymer polyhydroxybutyrate (PHB). By creating in-frame fusions of phasins and green fluorescent protein (GFP) as a model protein, we demonstrated that GFP can be efficiently sequestered to the surface of PHB granules. In a second step, we generated a phasin-intein-GFP fusion, wherein the self-cleaving intein can be activated by the addition of thiols. This construct allowed for the controlled binding and release of essentially pure GFP in a single separation step. Finally, pure, active beta-galactosidase was obtained in a single step using the above described method.
Barnard
Gavin C
GC
Thayer School of Engineering, Department of Biological Sciences, and Department of Chemistry, Dartmouth College, 8000 Cummings Hall, Hanover, NH 03755, USA.
McCool
Jesse D
JD
Wood
David W
DW
Gerngross
Tillman U
TU
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001709:Biotechnology / Q000379:methods; D020118:Cupriavidus necator / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004229:Dithiothreitol; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006885:Hydroxybutyrates / Q000378:metabolism; D047668:Inteins / Q000235:genetics; D011091:Polyesters / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D006885:Hydroxybutyrates; C118828:PHAP protein, Bacteria; D011091:Polyesters; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004229:Dithiothreitol