Polymeric And Affinity Ligands For Protein Chromatography

The exploitation of novel ligands for protein purification and the further advancement of our understanding of bioseparation principles are crucial for the development of bioseparation engineering and technology, as well as for the improvement of separation processes of biotechnology products. This dissertation focused on the design, development and application of polymeric and affinity ligands, and details of this work are summarized as follows.In the first place, the present work developed a polymeric ligand, poly(4-vinylpyridine)(P4VP), for mixed-mode chromatography of proteins. In addition to the hydrophobic nature, P4 VP modified Sepharose resins also possess electrostatic interaction properties. The mixed-mode adsorbent with immobilized P4 VP showed pH dependent and salt tolerant properties for protein binding with γ-globulin and BSA as model proteins. Electrostatic interaction was found to be a dominant contributor to protein adsorption, while hydrophobic interaction also worked to provide the salt-tolerant behavior in protein adsorption. The inverse size-exclusion chromatography(iSEC) experiment revealed that P4 VP formed a three-dimensional layer on the matrix surface with a maximum layer depth of 4.2 nm, which could significantly improve the binding capacity of proteins. Efficient protein recovery was achieved at mild elution conditions. The results indicate that the P4VP-based adsorbent would provide new possibilities for protein purification by MMC.To further investigate the properties and application of polymeric ligand, six PAA-modified Sepharose FF resins with different ionic capacities(ICs) were synthesized. Adsorption equilibria, kinetics and recovery experiments were studied under different ionic capacities and ionic strength with BSA as model protein. An obvious change was found at IC=373 mmol/L, above which the adsorption capacity and effective protein diffusivity(De) increased sharply. Moreover, the adsorption performance of PAA-modified resins exhibited less sensitive to salt concentration than PEI-modified resins and the commercial non-grafting resins. Recovery experiments found that a mild condition of pH 5.0 was sufficient for the effective elution of bound proteins with an overall protein recovery above 90%. The results indicate that PAA is a promising ligand for protein separation and purification.For the high efficient purification of murine polyomavirus virus-like particle(VLP), a biomimetic design strategy of affinity peptide ligand of capsomere(Cap) has been established on the basis of a binding structure of the C-terminus of minor coat protein(VP2-C) on the inner surface of Cap. The molecular interactions between VP2-C and Cap were first examined using all-atom molecular dynamics(MD) simulations coupled with the molecular mechanics-Poisson-Boltzmann surface area(MM/PBSA) method, where V283, P285, D286, W287, L289, and Y296 of VP2-C are identified as the hot spots. An affinity peptide library(DWXLXLXY) was then constructed for sequent virtual screening by docking with AUTODOCK VINA, binding structure comparison, and final docking with ROSETTA Flex PepDock, where ten peptide candidates were selected and further validated by MD simulations and MM/PBSA. DWDLRLLY was then experimentally validated by one-step purification of Cap from crude cell lysate using affinity chromatography with immobilized DWDLRLLY. Therefore, it is expected that the biomimetic design strategy would greatly facilitate the production and application of VLP.