High-Throughput Recovery of Therapeutic Proteins from the Inclusion Bodies of Escherichia coli

Abstract

Escherichia coli is widely used for the expression of recombinant proteins that do not require glycosylation for their bioactivity (1,2). Many commercially available recombinant hormones and the majority of interleukins and interferons are all expressed and produced in E. coli systems (3). The advantages of using E. coli as an expression system include the enormous volume of data on its cell biology, its fermentation process development, and its ability to produce relatively large and inexpensive quantities of recombinant protein (4). However, the high-level expression of recombinant proteins in E. coli often results in the accumulation of the product as insoluble aggregates in vivo as inclusion bodies (5). Inclusion body proteins are devoid of biological activity and require elaborate solubilization and refolding procedures to recover functional activity (6,7). The renaturation of inclusion body proteins into a bioactive form is cumbersome, results in low recovery of the final product, and also accounts for the major cost in overall production of recombinant proteins using E. coli (7,8). However, whereas high-yielding processes are developed for the refolding of the aggregated recombinant proteins, high-level expression of proteins as inclusion bodies provides a straightforward strategy for producing therapeutic proteins. The initial high level of expression compensates for loss during recovery of the protein of interest from inclusion bodies. Despite many successful refolding procedures for inclusion body proteins (even at industrial scale), the renaturation process for each protein is quite different. Most often, this process is carried out in an empirical way and causes poor recovery of the bioactive therapeutic protein.