Efficient production of human mitochondrial chaperonin (Hsp60/Hsp10) in Escherichia coli using a polyprotein strategy.

Escherichia coli remains the leading platform for recombinant protein production, yet co-expression of multiprotein complexes often suffers from stoichiometric imbalance, presenting a persistent challenge to precise subunit assembly. Many important biological functions are performed not by single proteins but by multiprotein complexes. Studying the structure and function of multiprotein complexes would be greatly facilitated if they could be reliably overproduced in heterologous host organisms. Here, we describe a simple method for the production of the human mitochondrial chaperonin (Hsp60/Hsp10) in Escherichia coli. Rather than producing the two chaperonin subunits from a polycistronic mRNA, a strategy commonly employed by bacterial operons, we chose to make them in the form of a single polyprotein that is subsequently cleaved inside bacterial cells by tobacco vein mottling virus (TVMV) protease. In this way, equimolar amounts of mature Hsp60 and Hsp10 subunits could be ensured. The TVMV protease is produced from a second mRNA that is transcribed from the same plasmid. Although expressed at a much lower level than the polyprotein, enough TVMV protease is produced to cleave all of the Hsp10/Hsp60 fusion protein in vivo. Moreover, we show that the mitochondrial chaperonin is fully functional when produced in this manner.