Application Of Polyhydroxybutyrate Granule In Recombinant Human Tissue-type Plasminogen Activator (rPA) Expression

In certain bacteria, polyhydroxyalkanoates (PHAs) are synthesized as a carbon and energy reserve material in times of unbalanced nutrient availability like excessive carbon resource or limitation of other essential nutrient. They are naturally produced from renewable resources and can be degraded by a variety of microorganisms, having many putative industrial and medical application with nontoxicity, good thermoplasticity and elasticity. Polyhydroxybutyrate (PHB) is the represented and the earliest known PHASCL.They are deposited as insoluble nano/micro-particles in cytoplasm, composed of a hydrophobic, amorphous polymer core surrounded by a monolayer of phospholipids with attached proteins, among which PhaP cover most of the granule surface. More recently, PHB has been increasingly used in biomedical fields for purifying proteins and targeted drug delivery at the granule surface.In the study, three enzymes,β-ketothiolase (PhbA), stereo-specific reductase (PhbB), and PHA synthase (PhbC) from Ralstonia eutropha H16 are necessary for PHB biosynthesis and were placed under the rpoS promoter to form a stress-induced PHB operon, by which the PHB biosynthesis pathways can be induced under stress conditions. The plasmid pHBS01 with PHB operon was transformed into Eschericha. coli XL1-Blue and BL21(DE3), respectively. The recombinant E. coli strains were cultivated in LB medium with 0.5% glucose. Results showed that the final biomass of XL1-blue/pHBS01 was nearly twice as much as that of BL21(DE3)/pHBS01, with 25% PHB of the cell dry weight, whereas BL21(DE3)/pHBS01 accumulated only about 9% (w/w). Moreover, the PHB granules size in XL1-blue was relatively small and well-proportioned.PhaP is the major PHB-specific binding protein. The desired proteins can bind to the PHB granules via the PHB-binding tag. The granules with attached proteins can then be easily recovered following cell lysis by ultracentrifugation. Comparing with traditional protein purification strategies, this method is cost-effective and reliable. So E. coli XL1-Blue/pHBS01 is the PHB-granule based protein expression system.Under physiological conditions, the reductive cytoplasmic environment of wide-type E. coli is the bottleneck of the oxidative folding of heterologous proteins with multiple disulfide bonds, which. Heterologous expression in E. coli often results in the formation of the insoluble aggregates known as inclusion bodies. So we chose rPA as the targeted protein. rPA contains nine disulfide bonds and is difficult to refolding in vitro. rPA was fused to the N-terminus of PhaP protein with a thrombin cleavage site as the linker. The fusion gene was placed under Plac to form the plasmid pSPAr. Then pSPAr was transformed into XL1-Blue/pHBS01. The recombinant was cultivated in LB 0.5% glucose and PhaP-rPA fusion was induced at late exponential phase. SDS-PAGE and immunoblot analysis showed that rPA fusion was successfully displayed on the surface of PHB granules. Activity assay indicated that the rPA fusion is active, reaching 760 IU/g cdw (cell dry weight). According to standard rPA, we assessed the yield of rPA was about 53.8 g/g cells. The results showed that the biosynthesis of PHB granules in vivo is a NADPH-dependent pathway and therefore may effectively promote the disulfide bonds formation. In addition, PHB granules are highly stable and the purification is simple and cost-effective.In the study, the PHB expression system is a novel method for the heterologous expression and purification of proteins with disulfide bonds.In the study, we used double plasmids system for rPA displaying on the PHB granules surface, which brought host cells metabolic burden and may limit the application of the PHB-based expression system. Therefore, the three enzymes for PHB biosynthesis were placed under the modified Ptac to form a PHB operon, which was then integrated into the poxB gene site on E. coli genome. In M9 medium with glucose, the recombinant strain can accumulate PHB spontaneously. In order to increase the yield, we replaced the sigle copy promoter Ptac with multicopy PMtac. Results indicated that the yield was increased five times, which offers theoretical induction for further genetic manipulation and technological base for PHB-based expression system.