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Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris.

Haon M, Grisel S, Navarro D, Gruet A, Berrin JG, Bignon C - Front Microbiol (2015)

Bottom Line: We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol.Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed.The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1163 Biodiversité et Biotechnologie Fongiques Marseille, France ; Aix-Marseille Université, Polytech Marseille, UMR1163 Biodiversité et Biotechnologie Fongiques Marseille, France.

ABSTRACT
Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community.

No MeSH data available.


Related in: MedlinePlus

Pichia pastoris recombinant protein expression platform. Automated purification by affinity chromatography on Ni-NTA and enzymatic activities assays of His6-tagged recombinant proteins are boxed. See Section “Materials and Methods” for details.
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Figure 5: Pichia pastoris recombinant protein expression platform. Automated purification by affinity chromatography on Ni-NTA and enzymatic activities assays of His6-tagged recombinant proteins are boxed. See Section “Materials and Methods” for details.

Mentions: Since deepwell plates can be directly transferred from a shaking incubator to a robot benchwork, the recovery of His6-tagged recombinant proteins by affinity chromatography on Ni-NTA was automated. To that end, Ni-NTA beads were used in deepwell filter plates as described in Section “Materials and Methods.” Note that 100 μl of beads per well were used because using 50 μl provided less reproducible results (not illustrated). The flowchart of the automated process is illustrated by Figure 5, and a thorough description of the robot can be found in (Navarro et al., 2010). After elution, recombinant His6-tagged proteins were analyzed by gel electrophoresis using pre-cast gels, and by assaying their enzymatic activity (Figure 6).


Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris.

Haon M, Grisel S, Navarro D, Gruet A, Berrin JG, Bignon C - Front Microbiol (2015)

Pichia pastoris recombinant protein expression platform. Automated purification by affinity chromatography on Ni-NTA and enzymatic activities assays of His6-tagged recombinant proteins are boxed. See Section “Materials and Methods” for details.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585289&req=5

Figure 5: Pichia pastoris recombinant protein expression platform. Automated purification by affinity chromatography on Ni-NTA and enzymatic activities assays of His6-tagged recombinant proteins are boxed. See Section “Materials and Methods” for details.
Mentions: Since deepwell plates can be directly transferred from a shaking incubator to a robot benchwork, the recovery of His6-tagged recombinant proteins by affinity chromatography on Ni-NTA was automated. To that end, Ni-NTA beads were used in deepwell filter plates as described in Section “Materials and Methods.” Note that 100 μl of beads per well were used because using 50 μl provided less reproducible results (not illustrated). The flowchart of the automated process is illustrated by Figure 5, and a thorough description of the robot can be found in (Navarro et al., 2010). After elution, recombinant His6-tagged proteins were analyzed by gel electrophoresis using pre-cast gels, and by assaying their enzymatic activity (Figure 6).

Bottom Line: We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol.Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed.The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1163 Biodiversité et Biotechnologie Fongiques Marseille, France ; Aix-Marseille Université, Polytech Marseille, UMR1163 Biodiversité et Biotechnologie Fongiques Marseille, France.

ABSTRACT
Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community.

No MeSH data available.


Related in: MedlinePlus