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Rapid Matrix‐Assisted Refolding of Histidine‐Tagged Proteins
Author(s) -
Dashivets Tetyana,
Wood Nichole,
Hergersberg Christoph,
Buchner Johannes,
Haslbeck Martin
Publication year - 2009
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200800697
Subject(s) - inclusion bodies , protein folding , chemistry , folding (dsp implementation) , recombinant dna , matrix (chemical analysis) , protein aggregation , monomer , biochemistry , biophysics , chromatography , biology , polymer , organic chemistry , gene , engineering , electrical engineering
Matrix refolded : The formation of inclusion bodies, which are amorphous aggregates of misfolded insoluble protein, during recombinant protein expression, is one of the biggest bottlenecks in protein science. We report a stepwise, rational optimization procedure for refolding of insoluble proteins (see scheme). In comparison to refolding in‐solution, this parallelized, matrix‐assisted approach allows the refolding of various proteins in a fast and efficient manner.The formation of inclusion bodies (IBs)—amorphous aggregates of misfolded insoluble protein—during recombinant protein expression, is still one of the biggest bottlenecks in protein science. We have developed and analyzed a rapid parallel approach for matrix‐assisted refolding of recombinant His 6 ‐tagged proteins. Efficiencies of matrix‐assisted refolding were screened in a 96‐well format. The developed methodology allowed the efficient refolding of five different test proteins, including monomeric and oligomeric proteins. Compared to refolding in‐solution, the matrix‐assisted refolding strategy proved equal or better for all five proteins tested. Interestingly, specifically oligomeric proteins displayed significantly higher levels of refolding compared to refolding in‐solution. Mechanistically, matrix‐assisted folding seems to differ from folding in‐solution, as the reaction proceeds more rapidly and shows a remarkably different concentration dependence—it allows refolding at up to 1000‐fold higher protein concentration than folding in‐solution.