Strategies for boosting the accumulation of correctly folded recombinant proteins expressed in Escherichia coli

The yields of soluble recombinant protein expressed in bacteria can be significantly enhanced by optimally exploiting the cell-folding machinery. The proposed protocol describes the strategies that can be used to reach a suitable ratio between heat-shock proteins and target proteins. Specifically, molecular recombinant chaperones can be overexpressed or cell-native chaperones are stimulated by inducing chemical and physical stress. Furthermore, the protein synthesis block can make available the cell-folding machinery for in vivo, disaggregating and refolding the already produced misfolded recombinant target protein. A rapid fluorimetric analytical method allows the evaluation of the protein monodispersity at any single purification step and enables comparison of different growth combinations that are useful to test for screening the optimal conditions for each recombinant protein.