Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli

Ario De Marco,Elke Deuerling,Axel Mogk,Toshifumi Tomoyasu,Bernd Bukau

doi:10.1186/1472-6750-7-32

Abstract

BackgroundThe overproduction of recombinant proteins in host cells often leads to their misfolding and aggregation. Previous attempts to increase the solubility of recombinant proteins by co-overproduction of individual chaperones were only partially successful. We now assessed the effects of combined overproduction of the functionally cooperating chaperone network of the E. coli cytosol on the solubility of recombinant proteins.ResultsA two-step procedure was found to show the strongest enhancement of solubility. In a first step, the four chaperone systems GroEL/GroES, DnaK/DnaJ/GrpE, ClpB and the small HSPs IbpA/IbpB, were coordinately co-overproduced with recombinant proteins to optimize de novo folding. In a second step, protein biosynthesis was inhibited to permit chaperone mediated refolding of misfolded and aggregated proteins in vivo. This novel strategy increased the solubility of 70% of 64 different heterologous proteins tested up to 42-fold.ConclusionThe engineered E. coli strains and the two-step procedure presented here led to a remarkable increase in the solubility of a various recombinant proteins and should be applicable to a wide range of target proteins produced in biotechnology.

Highlights

The overproduction of recombinant proteins in host cells often leads to their misfolding and aggregation
In the E. coli cytosol, the folding of newly synthesized proteins is assisted by the ribosome-associated Trigger Factor, the DnaK system (DnaK with its DnaJ and GrpE cochaperones; KJE), and the GroEL system
The co-overproduction of ELS increases the solubility of human ORP150, human lysozyme, p50csk protein tyrosine kinase, phosphomannose isomerase and fusion protein PreS2-S'-β-galactosidase and maize protoporphyrinogen IX oxidase [18,19,20,21,22,23]

Summary

Introduction

The overproduction of recombinant proteins in host cells often leads to their misfolding and aggregation. Previous attempts to increase the solubility of recombinant proteins by co-overproduction of individual chaperones were only partially successful. Chaperones assist the folding of newly synthesized proteins to the native state and provide a quality control system that refolds misfolded and aggregated proteins. In the E. coli cytosol, the folding of newly synthesized proteins is assisted by the ribosome-associated Trigger Factor, the DnaK system (DnaK with its DnaJ and GrpE cochaperones; KJE), and the GroEL system (GroEL with its GroES cochaperone; ELS) [1,2,3,4]. KJE cooperates with ClpB to solubilize aggregated proteins and refold them to the native state in a concerted action with ELS [7,8,9]. Several attempts were made to increase the yields of correctly folded, and soluble, recombinant proteins by the co-overproduction of individual chaperones in producing cells, only with a limited success. The co-overproduction of KJE increases the solubility of endostatin, human ORP150, transglutaminase and PreS2-S'-β-galactosidase [21,24,25]

Methods

Results

Conclusion