title: Single pH buffer refolding screen for protein from inclusion bodies.
abstract: We previously reported the set up of an automated test for screening the refolding of recombinant proteins expressed as inclusion bodies in Escherichia coli[1]. The screen used 96 refolding buffers and was validated with 24 proteins, 70% of which remained soluble in at least one buffer. In the present paper, we have analyzed in more detail these experimental data to see if the refolding process can be driven by general rules. Notably, we found that proteins with an acidic isoelectric point (pI) refolded in buffers the average pH of which was alkaline and conversely. In addition, the number of refolding buffers wherein a protein remained soluble increased with the difference between its pI and the average pH of the buffers in which it refolded. A trend analysis of the other variables (ionic strength, detergents, etc.) was also performed. On the basis of this analysis, we devised and validated a new refolding screen made of a single buffer for acidic proteins and a single buffer for alkaline proteins.
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 7257 CNRS and Aix-Marseille University, 163, Avenue de Luminy, Case 932, 13288 Marseille, Cedex 09, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Coutard
authors.forename: Etienne G J
authors.initials: EG
authors.lastname: Danchin
authors.forename: Rachid
authors.initials: R
authors.lastname: Oubelaid
authors.forename: Bruno
authors.initials: B
authors.lastname: Canard
authors.forename: Christophe
authors.initials: C
authors.lastname: Bignon
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D006801:Humans; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry*; D007202:Indicators and Reagents; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D058849:Protein Refolding; D020543:Proteome / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D007202:Indicators and Reagents; D020543:Proteome; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C493188:Rv1399c protein, M tuberculosis; C070196:feruloyl esterase