Background: In the current research, recombinant Cross-Reactive Material (rCRM 197) designed to form inclusion bodies in recombinant E. coli cells. High expression of recombinant proteins forms as aggregates in recombinant E. coli with misfolded and acts as inactive proteins. Regularly fermentation process is carried out at 37 °C to produce rCRM197 Inclusion bodies in recombinant E. coli, which forms very strong aggregates. As per researcher, reduction of temperature leads to less aggregates and more active refolded proteins can be formed. Inclusion bodies formed at low temperature called as non- classical Inclusion bodies.