Rehosting of Bacterial Chaperones for High-Quality Protein Production

Abstract

Coproduction of DnaK/DnaJ in Escherichia coli enhances solubility but promotes proteolytic degradation of their substrates, minimizing the yield of unstable polypeptides. Higher eukaryotes have orthologs of DnaK/ DnaJ but lack the linked bacterial proteolytic system. By coexpression of DnaK and DnaJ in insect cells with inherently misfolding-prone recombinant proteins, we demonstrate simultaneous improvement of soluble protein yield and quality and proteolytic stability. Thus, undesired side effects of bacterial folding modulators can be avoided by appropriate rehosting in heterologous cell expression systems.

Cross-system excision of chaperone-mediated proteolysis in chaperone-assisted recombinant protein production

“…1) has been proved by the production of a modified aggregationprone GFP (mGFP) along with DnaK and DnaJ in insect cells, where the chaperones compensate for the conformational stress during recombinant protein production in absence of proteolysis. 30 In fact, the recombinant GFP is proteolytically stabilized in the presence of DnaK and DnaJ, 30 contrarily to what had been observed in E. coli, where the half life of this protein is dramatically reduced under co-expression conditions. 18 Moreover, when analyzing protein fractioning in cultured cells, protein yield is significantly favoured in the soluble fraction and also in the insoluble fraction.…”

“…In agreement, in cells overexpressing DnaK and its immediate co-chaperone DnaJ along with the target recombinant protein, solubility increases with a concomitant loss of conformational quality and protein yield, being these parameters mutually exclusive. 18 In the context of the antagonistic activities of the DnaK and DnaJ pair observed in bacteria, the introduction of these prokaryotic folding modulators in heterologous expression systems such as cultured insect cells, 30 insect larvae 31 or others can benefit from their conserved foldase activities while avoiding the undesirable adverse proteolytic effect mediated by ClpP and Lon proteases, which is not expected to be conserved in eukaryotes. This principle (Fig.…”

“…18 Moreover, when analyzing protein fractioning in cultured cells, protein yield is significantly favoured in the soluble fraction and also in the insoluble fraction. When extending the chaperone rehosting concept to the production of other recombinant proteins in insect cell culture, these are better produced under co-production conditions than in absence of bacterial DnaK and DnaJ, 30 showing an increase in the yield of extractable protein and a reduction of oligomer formation depending on the model protein. 30 When rehosting the chaperones to a whole insect larvae system, total protein levels were not significantly affected by chaperone coproduction, but soluble protein amounts represented almost the total of the recombinant protein and solubility values higher than 90% were obtained.…”

Co-expression of chaperones from P. furiosus enhanced the soluble expression of the recombinant hyperthermophilic α-amylase in E. coli

“…Molecular chaperones have diverse biological functions such as protecting cells from adverse environmental conditions, catalyzing solubility and refolding of stable protein aggregates (Ben-Zvi and Goloubinoff 2001), assisting folding of the newly synthesized polypeptide (Hartl and Hayer-Hartl 2002), and allowing the evolution of new protein functions and phenotypic traits (Tokuriki and Tawfik 2009;Rutherford and Lindquist 1998;Hartl et al 2011). These functions render chaperones as promising candidates for biotechnological applications (de Marco 2007;Martinez-Alonso et al 2009). …”

“…de Marco (2007) co-expressed eight combinations of E. coli molecular chaperones with a target protein in order to improve target protein's soluble yield. Martinez-Alonso et al (2009) achieved high-quality recombinant protein production by rehosting bacterial chaperones in eukaryotes.…”