Co-expression of a heat shock transcription factor to improve conformational quality of recombinant protein in Escherichia coli

• Co-expression of heat shock transcription factor sigma 32 in recombinant Escherichia coli.
• Higher enzymatic activity of overexpressed protein in both soluble fraction and inclusion body.
• Promoting conformational quality of the recombinant protein by the co-expression of sigma 32.
• Co-expression led to a decrease in the particle size of inclusion bodies.

A co-expression system was established in Escherichia coli for enhancing the cellular expression of heat shock transcription factor, sigma 32 (σ32). A Shine–Dalgarno sequence and the rpoH gene of E. coli, which encodes σ32, were cloned into a bacterial plasmid containing a gene fusion encoding a doubly tagged N-acetyl-d-neuraminic acid aldolase (GST-Neu5Ac aldolase-5R). After the IPTG induction, a substantially higher level of sigma 32 was observed up to 3 h in the co-expression cells, but an enhancement in the solubility of target protein was manifest only in the first hour. Nevertheless, the co-expression of sigma 32 led to higher level of Neu5Ac aldolase enzymatic activity in both the soluble and insoluble (inclusion body) fractions. The Neu5Ac aldolase activity of the supernatant from the lysate of cells co-expressing GST-Neu5Ac aldolase-5R and recombinant σ32 was 3.4-fold higher at 3 h postinduction than that in cells overexpressing GST-Neu5Ac aldolase-5R in the absence of recombinantly expressed σ32. The results of acrylamide quenching indicated that the conformational quality of the fusion protein was improved by the co-expression of recombinant σ32. Thus, the increased level of intracellular σ32 might have created favorable conditions for the proper folding of recombinant proteins through the cooperative effects of chaperones/heat shock proteins expressed by the E. coli host, which resulted in smaller inclusion bodies, improved conformational quality and a higher specific activity of the overexpressed GST-Neu5Ac aldolase-5R protein.