Abstract
Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of three-phase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding 12 different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP-refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Animals
-
Bacterial Proteins / biosynthesis
-
Bacterial Proteins / chemistry
-
Bacterial Proteins / isolation & purification
-
Bacterial Toxins / biosynthesis
-
Bacterial Toxins / chemistry
-
Bacterial Toxins / isolation & purification
-
CD4 Antigens / biosynthesis
-
CD4 Antigens / chemistry
-
CD4 Antigens / isolation & purification
-
Drosophila Proteins / biosynthesis
-
Drosophila Proteins / chemistry
-
Drosophila Proteins / isolation & purification
-
Escherichia coli / chemistry*
-
Escherichia coli / metabolism
-
Humans
-
Inclusion Bodies / chemistry*
-
Protein Folding
-
Protein Renaturation*
-
Protein Tyrosine Phosphatases / biosynthesis
-
Protein Tyrosine Phosphatases / chemistry
-
Protein Tyrosine Phosphatases / isolation & purification
-
Recombinant Proteins / biosynthesis
-
Recombinant Proteins / chemistry
-
Recombinant Proteins / isolation & purification*
-
Ribonuclease, Pancreatic / chemistry
-
Ribonuclease, Pancreatic / isolation & purification
Substances
-
Bacterial Proteins
-
Bacterial Toxins
-
CD4 Antigens
-
CcdB protein, Plasmid F
-
Drosophila Proteins
-
Recombinant Proteins
-
Ribonuclease, Pancreatic
-
PTP-ER protein, Drosophila
-
Protein Tyrosine Phosphatases