PMID:12182832
| Citation |
Rial, DV and Ceccarelli, EA (2002) Removal of DnaK contamination during fusion protein purifications. Protein Expr. Purif. 25:503-7 |
|---|---|
| Abstract |
The use of fusion proteins for recombinant protein expression in Escherichia coli has become popular because the carrier increases protein solubility, standardizes expression levels, and facilitates purification of the fusion products. However, we have observed that the peptide regions that fuse the carrier to the protein of interest bind E. coli Hsp70 molecular chaperones (DnaK) depending on their amino acid composition, resulting in an unwanted contamination during protein purification. Here we describe an approach that helps to circumvent this unwanted contamination. First, the appropriate amino acids surrounding and comprising the cloning site are chosen by using a software based on an algorithm already developed to decrease to a minimum the propensity of the fusion protein to bind DnaK. Second, DnaK contamination is significantly reduced by washing the fusion protein bound to the purification resin with MgATP plus soluble denatured E. coli proteins before elution. The approach can also be applied to eliminate other molecular chaperones. |
| Links | |
| Keywords |
Amino Acid Sequence; Bacterial Proteins; Escherichia coli/chemistry; Escherichia coli/genetics; Escherichia coli Proteins/analysis; Escherichia coli Proteins/isolation & purification; HSP70 Heat-Shock Proteins/analysis; HSP70 Heat-Shock Proteins/isolation & purification; Molecular Sequence Data; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/isolation & purification; Software |
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