Characterization of intermediate states of protein folding and obtainment of biofunctional proteins by high hydrostatic pressure

Abstract

Many relevant therapeutic proteins are found naturally in low concentrations. Escherichia coli is a very useful microorganism for recombinant protein production for structural and functional studies and for production of therapeutic proteins in high levels. However, the production of recombinant proteins often results in the accumulation of insoluble aggregates (inclusion bodies, IB) in bacterial cytoplasm, which is a serious problem for biotechnology because it decreased the yield of bioactive proteins. The application of high pressure is a mild technique for dissociation of protein aggregates which enables maintenance of secondary and tertiary structures, and can be useful for proteins refolding to increase yields of native proteins. In this project, we will develop models for using high pressure to promote protein refolding from aggregates to increase yields of natively active proteins. To this end, we will conduct studies of protein stability and characterization of intermediate protein states in the unfolding/folding pathways of human growth hormone (hGH) and cruzain. We will optimize the conditions of pressure, temperature and buffer for the acquisition of high levels of active native state using high hydrostatic pressure for each of the monomeric proteins Bcp, TrxA, TsnC and Ybbn of X. fastidiosa, PilB of X. axonopodis pv citri, SM29 from S. mansoni and GFP and the oligomeric proteins CTB and fiber knob of adenovirus. We will also study the cultivation conditions to optimize the production of easily dissociable IB of the recombinant proteins. (AU)