Minimizing inclusion body formation during recombinant protein production in Escherichia coli at bench and pilot plant scale

Many recombinant proteins partially aggregate into inclusion bodies during production in Escherichia coli in batch culture on defined medium. Production on complex medium, however, effectively prevented inclusion body formation of a beta-galactosidase-HIVgp41 fusion protein for detection of anti-HIV antibodies which is produced at 42 degreesC under control of a temperature-inducible expression system. Cells pre-conditioned by cultivation on complex medium before induction showed faster growth, higher product concentration and reduced inclusion body formation even when producing on defined medium. In contrast, for human basic fibroblast growth factor (hFGF-2) produced under control of the phage T7-promoter, medium composition could not reduce inclusion body formation even at 30 degreesC. Here, slow production in high-cell density fed-batch mode using a defined medium with limited glucose feeding enabled the accumulation of 50 mg product per gram cell dry mass exclusively in the soluble cell fraction, resulting in a volumetric concentration of more than 4 g per litre hFGF-2. With the beta-galactosidase fusion protein produced in fed-batch, over 100 mg of product per gram cell dry mass accumulated in the soluble cell fraction. With a cell density of 100 g cell dry mass per hire, this resulted in a volumetric concentration of 10 g per litre of soluble beta-galactosidase-HIVgp41 fusion protein. Thus, two approaches to balance heterologous protein production and host physiology are presented, which fit the needs of lab bench or pilot plant, respectively. (C) 2003 Elsevier Inc. All tights reserved.