Integrated recombinant protein expression and purification platform based on Ralstonia eutropha

Abstract

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.

FIG. 1.

Representative GFP localization. Fluorescence microscopy and sucrose density gradient fractionation of cell lysates. a) Wild-type R.eutropha. b) R. eutropha G (expressing GFP). c) R. eutropha PG (expressing PhaP-GFP). d) R. eutropha PIG (expressing PhaP-intein-GFP). Cell lysates were generated by sonication and added to the top of a sucrose density gradient. Fluorescence of individual sucrose density gradient fractions is expressed in relative fluorescence units divided by 100 and shown in green. Corresponding PHB concentrations are expressed in mg/liter and shown in tan.

FIG. 2.

Intein mediated cleavage of GFP and beta-galactosidase from whole cell debris. R. eutropha strains were lysed by sonication, the supernatant discarded and the insoluble pellet containing PHB granules retained. Intein mediated cleavage was activated by incubating the washed pellet overnight in buffer B2 at 37°C. After incubation, the pellet and supernatant fractions were isolated. (a) Fluorometry. Green bars show the fluorescence of the supernatant fractions. Tan colored bars denote the fluorescence of the resulting pellet fraction. (b) SDS-PAGE of fractions. Lanes 2 and 3: R. eutropha wild-type pellet and supernatant following dithiothreitol treatment, respectively. Lanes 4 and 5: R. eutropha G pellet and supernatant. Lanes 6 and 7: R. eutropha PG pellet and supernatant. Lanes 8 and 9: R. eutropha PIG pellet and supernatant. (c) SDS-PAGE. Lane 2: R. eutropha PIL supernatant.

FIG.3.

Flowchart of a recombinant protein purification scheme based on PhaP mediated PHB granule sequestration. Ralstonia eutropha recombinants expressing phasin-intein-GFP (PIG) are cultivated in a shake flask or a bioreactor. Cells are harvested by centrifugation, washed and resuspended in buffer B1 prior to cell disruption. The cell lysate is centrifuged, the supernatant fraction discarded and the insoluble fraction retained. After washing the insoluble pellet, the fraction is resuspended in buffer B2. The intein is thiol activated and GFP is released from the whole cell debris into the supernatant. The pure protein is recovered by centrifugation, discarding the pellet and retaining the supernatant fraction.