Automated Analysis of Protein Folding

The ability to define and understand pathways for folding recombinant proteins can often be a rate-limiting step in the preparation of such proteins for the use in diagnostic tests, drug screening, or for structural analysis by NMR and X-ray crystallography. This chapter describes modifications to a standard HPLC system that will permit its use in automation of the analysis of protein folding. In this analysis, urea concentration is also manually determined in each sample using refractive index measurements, and urokinase activity is measured using a spectrophotometric kinetic titerplate reader from molecular devices. The system presented in the chapter has a number of pertinent advantages. With an integrated HPLC-triggered fraction collector and larger bore delay tubing the system can be set up to repetitively inject and collect preparative samples of unfolded protein to be refolded and analyzed. Because, the preparatively folded samples of protein will only be folded and active in a certain range of denaturant concentration, this approach permits recapture of misfolded material and recycling through the system.