Isolation, Solubilization, Refolding, and Chromatographic Purification of Human Growth Hormone from Inclusion Bodies of Escherichia coli Cells: A Case Study

Inclusion bodies produced in Escherichia coli are composed of densely packed denatured protein molecules in the form of particles ( 1 , 2 ). In addition to the recombinant protein of interest, inclusion bodies contain small amounts of host protein, ribosomal components, and DNA/RNA fragments ( 3 ). It is advisable to purify the inclusion bodies from the cells to a high-degree purity before carrying out solubilization and purification. This will reduce the number of purification steps after solubilization and refolding, minimize the interference of other contaminating proteins during refolding, and result in a therapeutic protein free from other cellular contaminants, such as lipids, carbohydrate, and endotoxin ( 4 ). Isolation of inclusion bodies from E. coli occurs by cell lysis with high-pressure disruption using a French press or sonication step followed by centrifugation ( 5 ). Further purification can be achieved by washing with detergents and a low concentration of salt and/or urea ( 5 , 6 ). The presence of contaminants, along with the protein of interest, is mainly because of incomplete purification of the inclusion bodies following cell lysis. With proper centrifugation and washing processes, more than 95% pure inclusion bodies of recombinant proteins can be isolated from E. coli cells ( 7 ). As the inclusion bodies have a high density (approx 1.3 mg mL −1 ), these are easily separated by high-speed centrifugation after cell disruption ( 8 ). Centrifugal isolation, particularly sucrose gradient centrifugation, has been found to be the best method for isolating very pure inclusion bodies from E. coli cell lysate ( 9 ). Expression of recombinant human growth hormone (r-hGH) in E. coli will illustrate the methods used for isolation and purification of intact inclusion bodies. The purified inclusion bodies will then be used for solubilization and refolding to obtain bioactive protein. These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.