Rapid Matrix-Assisted Refolding of Histidine-Tagged Proteins

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Abstract

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.

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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 His6-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.

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