Introduction

Protein expression in E. coli is an efficient and commonly used method to generate large quantities of protein for research or therapeutic applications. Unfortunately, proteins expressed at high levels in E. coli are often packaged into inclusion bodies (IBs). These tightly-packed structures have the advantage of being composed of almost pure expressed protein, but the serious disadvantage that the protein is so tightly aggregated that high concentrations of chaotropes or detergents are required to extract soluble protein from the aggregates. These solubilization reagents must then be diluted or removed by buffer exchange, so that the extracted protein can be refolded into its native, functional conformation.

High hydrostatic pressure has shown promise as a means of disaggregating and solubilizing protein aggregates using relatively mild buffer conditions [1-4]. By disaggregating IBs without the high levels of denaturants required under conventional conditions, subsequent protein refolding can be improved.

Here we report that high hydrostatic pressure can be used to efficiently disaggregate proinsulin inclusion bodies in order to extract soluble proinsulin protein. This disaggregation can be carried out in mild buffer conditions at ambient temperature in as little as 5 minutes at 45kpsi.