PMID:17948006
| Citation |
de Marco, A (2007) Protocol for preparing proteins with improved solubility by co-expressing with molecular chaperones in Escherichia coli. Nat Protoc 2:2632-9 |
|---|---|
| Abstract |
Eight combinations of molecular chaperones (e.g., DnaK/DnaJ/GrpE/ClpB) are co-expressed with the target recombinant protein to compare their effectiveness in improving its soluble yield. This system allows the most complete and rational approach proposed so far to use the chaperone activity for optimizing the host cell folding machinery. Furthermore, a two-step protocol is presented, in which protein synthesis and protein refolding are uncoupled. Molecular chaperones and target protein accumulate in the first growth phase and target protein aggregates are then disaggregated in vivo after the block of protein synthesis. The optimal chaperone combination to maximize the soluble yield of a specific protein remains unpredictable. Therefore, a small-scale purification selection step is useful for screening among expression combinations before scaling-up production. Applying such a strategy, we could increase the solubility of 70% of the tested constructs with yields of up to 42-fold more protein than in controls. The procedure takes 2 d. |
| Links |
PubMed Online version:10.1038/nprot.2007.400 |
| Keywords |
Bacteriological Techniques; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/isolation & purification; Escherichia coli Proteins/metabolism; Molecular Chaperones/genetics; Molecular Chaperones/metabolism; Protein Folding; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Solubility; Transformation, Genetic |
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