Sources and upstream processing - Pharmaceutical Biotechnology: Concepts and Aapplications - page 110

Figure 5.1 High-level expression of a protein of interest in E. coli in soluble form by using the engineered
'thiofusion' expression system. Refer to text for specifi c details

Various attempts have been made to prevent inclusion body formation when expressing heter-
ologous proteins in E. coli. Some studies have shown that a simple reduction in the temperature of
bacterial growth (from 37 C) can signifi cantly decrease the incidence of inclusion body

formation. Other studies have shown that expression of the protein of interest as a fusion partner with
thioredoxin will eliminate inclusion body formation in most instances. Thioredoxin is a homolo-
gous E. coli protein, expressed at high levels. It is localized at the adhesion zones in E. coli and is a
heat-stable protein. A plasmid vector has been engineered to facilitate expression of a fusion protein
consisting of thioredoxin linked to the protein of interest via a short peptide sequence recognized by
the protease enterokinase ( Figure 5.1). The fusion protein is invariably expressed at high levels, while
remaining in soluble form. Congregation at adhesion zones facilitates its selective release into the me-
dia by simple osmotic shock. This can greatly simplify its subsequent purifi cation. After its release,
the fusion protein is incubated with enterokinase, thus releasing the protein of interest ( Figure 5.1).

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