PMID- 20851186
OWN - NLM
STAT- MEDLINE
DCOM- 20110228
LR  - 20111117
IS  - 1096-0279 (Electronic)
IS  - 1046-5928 (Linking)
VI  - 75
IP  - 2
DP  - 2011 Feb
TI  - A high-throughput protein refolding screen in 96-well format combined with design 
      of experiments to optimize the refolding conditions.
PG  - 192-203
LID - 10.1016/j.pep.2010.09.008 [doi]
AB  - 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-1alpha/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.
CI  - Copyright (c) 2010 Elsevier Inc. All rights reserved.
FAU - Dechavanne, Vincent
AU  - Dechavanne V
AD  - Geneva Research Center, Merck Serono S.A., Department of Protein and Cell 
      Sciences, 9 Chemin des Mines, 1202 Geneva, Switzerland. 
      vincent.dechavanne@merckserono.net
FAU - Barrillat, Nicolas
AU  - Barrillat N
FAU - Borlat, Frederic
AU  - Borlat F
FAU - Hermant, Aurelie
AU  - Hermant A
FAU - Magnenat, Laurent
AU  - Magnenat L
FAU - Paquet, Mikael
AU  - Paquet M
FAU - Antonsson, Bruno
AU  - Antonsson B
FAU - Chevalet, Laurent
AU  - Chevalet L
LA  - eng
PT  - Journal Article
DEP - 20100917
PL  - United States
TA  - Protein Expr Purif
JT  - Protein expression and purification
JID - 9101496
RN  - 0 (Anaphylatoxins)
RN  - 0 (CXCL13 protein, human)
RN  - 0 (Chemokine CXCL12)
RN  - 0 (Chemokine CXCL13)
RN  - 0 (IL17F protein, human)
RN  - 0 (Interleukin-17)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Reducing Agents)
RN  - 143011-72-7 (Granulocyte Colony-Stimulating Factor)
SB  - IM
MH  - Anaphylatoxins/*chemistry/genetics/isolation & purification/*metabolism
MH  - Biological Assay
MH  - Chemokine CXCL12/*chemistry/genetics/isolation & purification/*metabolism
MH  - Chemokine CXCL13/*chemistry/genetics/isolation & purification/*metabolism
MH  - Cloning, Molecular
MH  - Escherichia coli
MH  - Granulocyte Colony-Stimulating Factor/*chemistry/genetics/isolation & 
      purification/*metabolism
MH  - *High-Throughput Screening Assays
MH  - Humans
MH  - Inclusion Bodies/*chemistry/metabolism
MH  - Interleukin-17/*chemistry/genetics/isolation & purification/*metabolism
MH  - Protein Renaturation
MH  - Recombinant Proteins/*chemistry/genetics/isolation & purification/*metabolism
MH  - Reducing Agents/chemistry/metabolism
MH  - *Research Design
EDAT- 2010/09/21 06:00
MHDA- 2011/03/01 06:00
CRDT- 2010/09/21 06:00
PHST- 2010/07/21 00:00 [received]
PHST- 2010/09/07 00:00 [revised]
PHST- 2010/09/12 00:00 [accepted]
PHST- 2010/09/21 06:00 [entrez]
PHST- 2010/09/21 06:00 [pubmed]
PHST- 2011/03/01 06:00 [medline]
AID - S1046-5928(10)00256-1 [pii]
AID - 10.1016/j.pep.2010.09.008 [doi]
PST - ppublish
SO  - Protein Expr Purif. 2011 Feb;75(2):192-203. doi: 10.1016/j.pep.2010.09.008. Epub 
      2010 Sep 17.