A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions.
Production of correctly folded and biologically active proteins in Escherichiacoli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions. After a first generic screen of 96 different refolding conditions the parameters that produced the best yield were further explored in a focused DOE-based screen. The refolding efficiency and the quality of the refolded protein were analyzed by RP-HPLC and SDS-PAGE. The results were analyzed by the DOE software to identify the optimal concentrations of the critical additives. The optimal refolding conditions suggested by DOE were verified in medium-scale refolding tests, which confirmed the reliability of the predictions. Finally, the refolded protein was purified and its biological activity was tested in vitro. The screen was applied for the refolding of Interleukin 17F (IL-17F), stromal-cell-derived factor-1 (SDF-1α/CXCL12), B cell-attracting chemokine 1 (BCA-1/CXCL13), granulocyte macrophage colony stimulating factor (GM-CSF) and the complement factor C5a. This procedure identified refolding conditions for all the tested proteins. For the proteins where refolding conditions were already available, the optimized conditions identified in the screening process increased the yields between 50% and 100%. Thus, the method described herein is a useful tool to determine the feasibility of refolding and to identify high-yield scalable refolding conditions optimized for each individual protein.
Dechavanne
Vincent
V
Geneva Research Center, Merck Serono S.A., Department of Protein and Cell Sciences, 9 Chemin des Mines, 1202 Geneva, Switzerland. vincent.dechavanne@merckserono.net
Barrillat
Nicolas
N
Borlat
Frederic
F
Hermant
Aurélie
A
Magnenat
Laurent
L
Paquet
Mikael
M
Antonsson
Bruno
B
Chevalet
Laurent
L
D000706:Anaphylatoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001681:Biological Assay; D054377:Chemokine CXCL12 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054382:Chemokine CXCL13 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry* / Q000378:metabolism; D020381:Interleukin-17 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019163:Reducing Agents / Q000737:chemistry / Q000378:metabolism; D012107:Research Design
D000706:Anaphylatoxins; C110663:CXCL13 protein, human; D054377:Chemokine CXCL12; D054382:Chemokine CXCL13; C440021:IL17F protein, human; D020381:Interleukin-17; D011994:Recombinant Proteins; D019163:Reducing Agents; D016179:Granulocyte Colony-Stimulating Factor