High-throughput automated refolding screening of inclusion bodies.
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.
Vincentelli
Renaud
R
Architecture et Fonction des Macromolécules Biologiques, UMR 6098, CNRS et Universités d'Aix-Marseille I et II, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Canaan
Stéphane
S
Campanacci
Valérie
V
Valencia
Christel
C
Maurin
Damien
D
Frassinetti
Frédéric
F
Scappucini-Calvo
Loréna
L
Bourne
Yves
Y
Cambillau
Christian
C
Bignon
Christophe
C
D002021:Buffers; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012371:Robotics / Q000295:instrumentation / Q000379:methods*
D002021:Buffers; D011994:Recombinant Proteins