Expression in Escherichia coli, refolding and crystallization of Aspergillus niger feruloyl esterase A using a serial factorial approach.
Hydrolysis of plant biomass is achieved by the combined action of enzymes secreted by microorganisms and directed against the backbone and the side chains of plant cell wall polysaccharides. Among side chains degrading enzymes, the feruloyl esterase A (FAEA) specifically removes feruloyl residues. Thus, FAEA has potential applications in a wide range of industrial processes such as paper bleaching or bio-ethanol production. To gain insight into FAEA hydrolysis activity, we solved its crystal structure. In this paper, we report how the use of four consecutive factorial approaches (two incomplete factorials, one sparse matrix, and one full factorial) allowed expressing in Escherichia coli, refolding and then crystallizing Aspergillus niger FAEA in 6 weeks. Culture conditions providing the highest expression level were determined using an incomplete factorial approach made of 12 combinations of four E. coli strains, three culture media and three temperatures (full factorial: 36 combinations). Aspergillus niger FAEA was expressed in the form of inclusion bodies. These were dissolved using a chaotropic agent, and the protein was purified by affinity chromatography on Ni column under denaturing conditions. A suitable buffer for refolding the protein eluted from the Ni column was found using a second incomplete factorial approach made of 96 buffers (full factorial: 3840 combinations). After refolding, the enzyme was further purified by gel filtration, and then crystallized following a standard protocol: initial crystallization conditions were found using commercial crystallization screens based on a sparse matrix. Crystals were then optimized using a full factorial screen.
Benoit
Isabelle
I
UMR 1163 INRA de Biotechnologie des Champignons Filamenteux, IFR86-BAIM, Université de Provence et de la Méditerranée, ESIL, 163 avenue de Luminy CP 925, 13288 Marseille cedex 09, France.
Coutard
Bruno
B
Oubelaid
Rachid
R
Asther
Marcel
M
Bignon
Christophe
C
D001234:Aspergillus niger / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D005656:Fungal Proteins; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase