Purification And Conjugation Of Pertactin:from Molecular Structure To Process Design

To overcome the difficulties in preparation of natural pertactin(nPRN)and recombinant pertactin(rPRN)for production of acellular pertussis vaccine(aPV),we developed a "dual-IEC”strategy for purification of nPRN from Bordetella pertussis and a flash-batch dilution method for refolding of rPRN inclusion bodies expressed in E.coli based on its structural characteristics.Besides,rPRN was conjugated with either diphtheria toxoid or tetanus toxoid.The resultant conjugates revealed an enhanced immunogenicity.The main innovations are as follows:(1)We performed the surface potential analysis with GRASP2 program for rPRN.The results demonstrated that there are two major charge patches,one negative and one positive,which are located separately on this linear protein.For this special feature,we designed a dual ion exchange chromatography(dual-IEC)strategy including an anionic exchange and a cationic exchange process for separation of natural pertactin from the heat extract of Bordetella pertussis.The initial anionic exchange chromatography concentrated the product from 1.7 to 14.6%,with recovery of 80%.The second cationic exchange chromatography increased the purity to 33%,with recovery of 83%.The final purification was accomplished by hydrophobic interaction chromatography,yielding a purity of 96%.The overall recovery of the three columns was 61%.(2)Recombinant pertactin was highly expressed in the form of inclusion bodies in E.coli.However,up to 75%of the protein turned out as aggregates when refolding by pulse-fed batch dilution.The conceivable route for aggregate formation was proposed as that the C-terminus of partially folded intermediate with a strong hydrophobic core would intertwine with that region of newly added denatured protein,resulting in aggregation between proteins with different folding states.The key factor for prevention of aggregate formation was to improve the synchronization of refolding.For this purpose,flash-batch dilution was conducted in a way of one-shoot feed and immediate mixing at a 5 L scale,achieving a refolding yield about 70%.The remaining aggregates were efficiently removed along with impurities by one-step chromatography of Ni-resin.The purity of monomeric pertactin was>98%.An overall yield was 320 mg per liter fermentation liquor with a total recovery of about 59%.(3)Conjugation of rPRN to diphtheria toxoid(DTd)or tetanus toxoid(TTd)was attempted to enhance the immunogenicity.The cross-linking reaction was mediated by EDC/sNHS in a mild way since the amino groups on the protein surface have been blocked as demonstrated by analyses of SDS-PAGE and pI titration.Separation of the conjugates from parent proteins was achieved by a one-step chromatography on Ni-resin.SDS-PAGE and high-performance size exclusion chromatography(HP-SEC)analyses showed the conjugates with>90%purity,among which mono-valent conjugates accounted for>65%.Characterization by circular dichroism and fluorescence displayed that the conjugates conserved an advanced structure of both PRN and the carrier proteins.The evaluation of the immunogenicity was performed on NIH mice.The results with respect to PRN-specific antibody levels,ex vivo cytokines release and GC B cell proliferation showed that conjugation of PRN to DTd led to improving mixed Thl/Th2 responses related to simply mixing,and that of PRN to TTd showed an increase mainly in the Th1 response.Besides,recombinant fimbriae serotype 3(fim3)was highly expressed in the form of inclusion bodies in E.coli.Optimized refolding condition was determined and yielded a refolding recovery of>50%.Purification was achieved through immobilized metal affinity chromatography combing size exclusion chromatography.A final product with a purity of>99%and an overall recovery of 30%were obtained,yielding about 33 mg rfim3 from 1L fennentation liquor.