The Process Fundamentals Of Surfactant Assisted Protein Refolding

The research into protein refolding techniques is of essential importance to the down stream processing of genetically engineered proteins. The experimental studies in this dissertation have shown an effective refolding of denatured lysozyme by dilution using the refolding buffer containing low concentration cetyltrimethyl- ammonium bromide (CTAB) and indicated its high potential in large scale processing. This dissertation was devoted to the comprehensive understanding of the process fundamentals of this novel refolding operation, using native hen egg white lysozyme as the protein sample. The interaction between protein and surfactant, the key to the formation and the dissociation of protein-surfactant complex, was investigated using various experimental and analytical procedures, as well as molecular simulation using simple lattice model with dynamic Monte Carlo simulation. The feasibility of this new refolding method was examined through its application to the refolding of recombinant human lysozyme and recombinant β-mannanase, in which other refolding methods were also tested. Chapter 1 started with a brief overview of the theoretical and experimental studies of protein refolding. Then a detailed summary of the refolding techniques was presented, in which the development of surfactant assisted protein refolding methods was highlighted due to its proven potential in large scale refolding. The problems and prospects of the research in this field were discussed. It was concluded that the formation and dissociation of protein-surfactant complex was the key to an effective refolding. It was thus the focus of studies on the refolding process fundamentals in terms of thermodynamics and kinetics. The latter should be based on both experimental studies using different protein samples and theoretical investigations centered on the molecular interaction between surfactant and protein and its implementations to protein structure. At the end of this chapter, a framework of the study presented in this dissertation was illustrated. Chapter 2 investigated the refolding of lysozyme by dilution with the refolding buffer containing low concentration CTAB. It was reported for the first time that the addition of CTAB in the refolding buffer led to a substantial increase in the refolding. The refolding yield was a function of the molar ratio of CTAB to lysozyme and the maximum yield was obtained at a ratio of 10. The changes of the surface tension of refolding solution indicated a rapid formation of CTAB-denatured lysozyme complex and a slow dissociation, upon the addition of redox reagents, i.e., GSSG/GSH, which led to the refolding to native lysozyme. The circular dichroism spectra analysis indicated the addition of CTAB led to enriched native-like secondary structures of lysoyzme, which favored the refolding. However the refolding was hindered once too many CTAB molecules were involved into the complex. Analysis of the refolding products by non-reductive SDS-PAGE and ion exchange chromatography showed the existence of native lysozyme, soluble aggregates and the CTAB-denatured lysozyme as major products in CTAB assisted refolding and the distribution of these products were determined by the molar ratio of CTAB to the denatured lysozyme. Chapter 3 presented the refolding of lysozyme using surfactant and β-cyclodextrin (β-CD), an established method known as artificial chaperone. It was shown that the charge properties of surfactant affected the refolding performance significantly. In case of CTAB and β-CD being used, the refolding was observed again as a function of the molar ratio of CTAB to denatured lysozyme and the optimal refolding was obtained at 10. A new operation scheme was developed in the present study, which greatly increased the refolding through the effective inhibition of the formation of protein aggregates and reduced the consumption of surfactant and β-CD. Analysis by SDS-PAGE showed the native lysozyme and insoluble aggregates as the major products of the refolding and the dist...