The pre-induction temperature affects recombinant HuGM-CSF aggregation in thermoinducible Escherichia coli

The overproduction of recombinant proteins in Escherichia coli leads to insoluble aggregates of proteins called inclusion bodies (IBs). IBs are considered dynamic entities that harbor high percentages of the recombinant protein, which can be found in different conformational states. The production conditions influence the properties of IBs and recombinant protein recovery and solubilization. The E. coli growth in thermoinduced systems is generally carried out at 30 degrees C and then recombinant protein production at 42 degrees C. Since the heat shock response in E. coli is triggered above 34 degrees C, the synthesis of heat shock proteins can modify the yields of the recombinant protein and the structural quality of IBs. The objective of this work was to evaluate the effect of different pre-induction temperatures (30 and 34 degrees C) on the growth of E. coli W3110 producing the human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) and on the IBs structure in a lambda pL/pR-cI857 thermoinducible system. The recombinant E. coli cultures growing at 34 degrees C showed a similar to 69% increase in the specific growth rate compared to cultures grown at 30 degrees C. The amount of rHuGM-CSF in IBs was significantly higher in cultures grown at 34 degrees C. Main folding chaperones (DnaK and GroEL) were associated with IBs and their co-chaperones (DnaJ and GroES) with the soluble protein fraction. Finally, IBs from cultures that grew at 34 degrees C had a lower content of amyloid-like structure and were more sensitive to proteolytic degradation than IBs obtained from cultures at 30 degrees C. Our study presents evidence that increasing the pre-induction temperature in a thermoinduced system allows obtaining higher recombinant protein and reducing amyloid contents of the IBs.

Automated summary

This study aimed to evaluate the effect of different pre-induction temperatures on the growth of E. coli and the structure of IBs. The results showed that E. coli cultures growing at 34 degrees Celsius had a higher specific growth rate and significantly higher levels of recombinant protein in the IBs, with lower amyloid-like structure content.