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High‐throughput automated refolding screening of inclusion bodies
Author(s) -
Vincentelli Renaud,
Canaan Stéphane,
Campanacci Valérie,
Valencia Christel,
Maurin Damien,
Frassinetti Frédéric,
ScappuciniCalvo Loréna,
Bourne Yves,
Cambillau Christian,
Big Christophe
Publication year - 2004
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.04806004
Subject(s) - structural genomics , inclusion bodies , protein folding , chemistry , buffer (optical fiber) , escherichia coli , solubility , throughput , computational biology , protein tag , computer science , chromatography , biochemistry , protein structure , recombinant dna , biology , telecommunications , organic chemistry , wireless , gene , fusion protein
One of the main stumbling blocks encountered when attempting to express foreign proteins in Escherichia coli is the occurrence of amorphous aggregates of misfolded proteins, called inclusion bodies (IB). Developing efficient protein native structure recovery procedures based on IB refolding is therefore an important challenge. Unfortunately, there is no “universal” refolding buffer: Experience shows that refolding buffer composition varies from one protein to another. In addition, the methods developed so far for finding a suitable refolding buffer suffer from a number of weaknesses. These include the small number of refolding formulations, which often leads to negative results, solubility assays incompatible with high‐throughput, and experiment formatting not suitable for automation. To overcome these problems, it was proposed in the present study to address some of these limitations. This resulted in the first completely automated IB refolding screening procedure to be developed using a 96‐well format. The 96 refolding buffers were obtained using a fractional factorial approach. The screening procedure is potentially applicable to any nonmembrane protein, and was validated with 24 proteins in the framework of two Structural Genomics projects. The tests used for this purpose included the use of quality control methods such as circular dichroism, dynamic light scattering, and crystallogenesis. Out of the 24 proteins, 17 remained soluble in at least one of the 96 refolding buffers, 15 passed large‐scale purification tests, and five gave crystals.