Role of the chaperone DnaK in protein solubility and conformational quality in inclusion body-forming Escherichia coli cells.
Misfolding-prone proteins produced in bacteria usually fail to adopt their native conformation and aggregate. In cells producing folding-reluctant protein species, folding modulators are supposed to be limiting, a fact that enhances protein deposition. Therefore, coproducing DnaK or other main chaperones along with the target protein has been a common approach to gain solubility, although with very inconsistent and often discouraging results. In an attempt to understand the reason for this inconsistency, the impact of exogenous DnaK (encoded in an accompanying plasmid) on two protein features observed as indicators of protein quality, namely solubility and functionality, has been analysed here through the specific fluorescence emission of a reporter Green Fluorescent Protein (GFP). Intriguingly, GFP solubility is strongly dependent on its own yield but poorly affected by DnaK levels. On the contrary, the specific fluorescence of both soluble and insoluble GFP populations is simultaneously modulated by the availability of DnaK, with a profile that is clearly dissimilar to that shown by protein solubility. Therefore, solubility, not being coincident with the biological activity of the target protein, might not be a robust indicator of protein quality.
Martínez-Alonso
Mónica
M
Institute for Biotechnology and Biomedicine, Department of Genetics and Microbiology and CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.
Vera
Andrea
A
Villaverde
Antonio
A
D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000737:chemistry / Q000502:physiology; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism / Q000502:physiology*; D002479:Inclusion Bodies; D017510:Protein Folding; D012995:Solubility
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D049452:Green Fluorescent Proteins; C063842:dnaK protein, E coli