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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

Expression results of different DNA constructs. (A–C), 25 μl of culture supernatant using the indicated expression plasmid were directly analyzed by SDS-PAGE. (D) GH11 from (B) and (C) was purified by affinity chromatography on Ni-NTA, and 25 μl of elution fraction were analyzed by SDS-PAGE. M, molecular weight markers. (A) 180, 130, 100, 70 (■), 55 (■), 40, 35, 25 (■), 15, 10 kDa. (B–D) 250, 150, 100, 75 (■), 50 (■), 37, 25 (■), 20, 10 kDa.
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Figure 1: Expression results of different DNA constructs. (A–C), 25 μl of culture supernatant using the indicated expression plasmid were directly analyzed by SDS-PAGE. (D) GH11 from (B) and (C) was purified by affinity chromatography on Ni-NTA, and 25 μl of elution fraction were analyzed by SDS-PAGE. M, molecular weight markers. (A) 180, 130, 100, 70 (■), 55 (■), 40, 35, 25 (■), 15, 10 kDa. (B–D) 250, 150, 100, 75 (■), 50 (■), 37, 25 (■), 20, 10 kDa.

Mentions: In a first trial, GH5, GH11, and GH45 coding sequences were sub-cloned by restriction/ligation (R/L) into episomal pBGP1 (Sasagawa et al., 2011) and integrative pGAPZαA and pPICZαA expression vectors as described in Section “Materials and Methods,” and recombinant expression was evaluated by SDS-PAGE in the supernatant of culture media. Using integrative vectors was expected to result in genetically stable P. pastoris strains as they are integrated into the yeast chromosome at the cost of more laborious screening, whereas an episomal vector could be isolated by a simple plasmid preparation procedure. In addition, episomal vectors have higher transformation efficiencies and more than one copy per cell. It was therefore of interest to assess whether these features were correlated to recombinant protein expression level. Results are reported in Figures 1A–C. Expression of GH5 and GH45 was plasmid-dependant: GH5 was better expressed by pPICZαA than by pGAPZαA whereas it was the opposite for GH45. No GH11 was visible in the culture medium of any plasmid condition. To check whether GH11 was expressed, it was purified by affinity chromatography on Ni-NTA as described in Section “Materials and Methods” and analyzed by SDS-PAGE (Figure 1D). GH11 was slightly better expressed by pPICZαA than by pGAPZαA, although at levels definitely lower than those of GH5 and GH45. On the basis of above results, integrative vectors pGAPZαA and pPICZ were chosen for expressing fungal CAZymes. Two attempts to make DNA constructs easier resulted in negative expression results: the use of Gateway cloning instead of R/L (Additional File 1), and the use of PCR to both generate and linearize integrative expression constructs in a single experiment (Liu et al., 2008; Yu et al., 2012) (Additional File 2). In conclusion, the classical R/L technique followed by linearization by restriction of expression integrative constructs before transformation was used in the rest of this study, as proposed by Invitrogen (easyselect_man.pdf).


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)

Expression results of different DNA constructs. (A–C), 25 μl of culture supernatant using the indicated expression plasmid were directly analyzed by SDS-PAGE. (D) GH11 from (B) and (C) was purified by affinity chromatography on Ni-NTA, and 25 μl of elution fraction were analyzed by SDS-PAGE. M, molecular weight markers. (A) 180, 130, 100, 70 (■), 55 (■), 40, 35, 25 (■), 15, 10 kDa. (B–D) 250, 150, 100, 75 (■), 50 (■), 37, 25 (■), 20, 10 kDa.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Expression results of different DNA constructs. (A–C), 25 μl of culture supernatant using the indicated expression plasmid were directly analyzed by SDS-PAGE. (D) GH11 from (B) and (C) was purified by affinity chromatography on Ni-NTA, and 25 μl of elution fraction were analyzed by SDS-PAGE. M, molecular weight markers. (A) 180, 130, 100, 70 (■), 55 (■), 40, 35, 25 (■), 15, 10 kDa. (B–D) 250, 150, 100, 75 (■), 50 (■), 37, 25 (■), 20, 10 kDa.
Mentions: In a first trial, GH5, GH11, and GH45 coding sequences were sub-cloned by restriction/ligation (R/L) into episomal pBGP1 (Sasagawa et al., 2011) and integrative pGAPZαA and pPICZαA expression vectors as described in Section “Materials and Methods,” and recombinant expression was evaluated by SDS-PAGE in the supernatant of culture media. Using integrative vectors was expected to result in genetically stable P. pastoris strains as they are integrated into the yeast chromosome at the cost of more laborious screening, whereas an episomal vector could be isolated by a simple plasmid preparation procedure. In addition, episomal vectors have higher transformation efficiencies and more than one copy per cell. It was therefore of interest to assess whether these features were correlated to recombinant protein expression level. Results are reported in Figures 1A–C. Expression of GH5 and GH45 was plasmid-dependant: GH5 was better expressed by pPICZαA than by pGAPZαA whereas it was the opposite for GH45. No GH11 was visible in the culture medium of any plasmid condition. To check whether GH11 was expressed, it was purified by affinity chromatography on Ni-NTA as described in Section “Materials and Methods” and analyzed by SDS-PAGE (Figure 1D). GH11 was slightly better expressed by pPICZαA than by pGAPZαA, although at levels definitely lower than those of GH5 and GH45. On the basis of above results, integrative vectors pGAPZαA and pPICZ were chosen for expressing fungal CAZymes. Two attempts to make DNA constructs easier resulted in negative expression results: the use of Gateway cloning instead of R/L (Additional File 1), and the use of PCR to both generate and linearize integrative expression constructs in a single experiment (Liu et al., 2008; Yu et al., 2012) (Additional File 2). In conclusion, the classical R/L technique followed by linearization by restriction of expression integrative constructs before transformation was used in the rest of this study, as proposed by Invitrogen (easyselect_man.pdf).

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