Limits...
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

Antibiotics have no effect on P. pastoris growth nor on protein expression. (A) Effect of antibiotics on cell growth. The y axis is the optical density of a 1/300 dilution of the culture. None, no antibiotic; Amp, 100 μg/ml ampicillin; Chlo, 34 μg/ml chloramphenicol; Kana, 50 μg/ml kanamycin; Tetra, 15 μg/ml tetracycline; Zeo, 100 μg/ml Zeocin. (B) Recombinant proteins purified by affinity chromatography on Ni-NTA beads from 4 ml culture. From 100 μl of elution volume, 15 μl were analyzed by SDS-PAGE along with 15 μl of non-purified supernatant (lanes S). M, molecular weight markers; from top to bottom: 116, 66.2, 45, 35, 25, 18.4, 14.4 kDa. Numbers 50, 100, 200 in lanes Chlo and Tetra indicate the volume in μl of 50% Ni-NTA beads suspension used. See results for details.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4585289&req=5

Figure 4: Antibiotics have no effect on P. pastoris growth nor on protein expression. (A) Effect of antibiotics on cell growth. The y axis is the optical density of a 1/300 dilution of the culture. None, no antibiotic; Amp, 100 μg/ml ampicillin; Chlo, 34 μg/ml chloramphenicol; Kana, 50 μg/ml kanamycin; Tetra, 15 μg/ml tetracycline; Zeo, 100 μg/ml Zeocin. (B) Recombinant proteins purified by affinity chromatography on Ni-NTA beads from 4 ml culture. From 100 μl of elution volume, 15 μl were analyzed by SDS-PAGE along with 15 μl of non-purified supernatant (lanes S). M, molecular weight markers; from top to bottom: 116, 66.2, 45, 35, 25, 18.4, 14.4 kDa. Numbers 50, 100, 200 in lanes Chlo and Tetra indicate the volume in μl of 50% Ni-NTA beads suspension used. See results for details.

Mentions: To check that antibiotics had really no effect on yeast growth, a clone of P. pastoris cells expressing GH5 from pGAPZαA was grown for 4 days in YPD in the presence or absence of the antibiotics listed above at concentrations used in laboratories to select resistant E. coli. Biomass was assessed by measuring the optical density of the culture at 600 nm at the end of that period, which definitely demonstrated the total innocuousness of this series of antibiotics (Figure 4A). The amount of recombinant protein secreted in the culture medium was next compared. To that end, His6-tagged GH5 was concentrated by affinity chromatography on Ni-NTA beads under different conditions that are detailed in the next paragraph. Results (Figure 4B) showed no difference between protein expression in the presence or absence of antibiotic, suggesting that antibiotics have no negative effect on the over expression of a recombinant protein in P. pastoris. In conclusion, if bacterial contaminations are an issue commonly used antibiotics can provide good protection at much lower cost than Zeocin, and without effect on P. pastoris cell growth or protein expression.


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)

Antibiotics have no effect on P. pastoris growth nor on protein expression. (A) Effect of antibiotics on cell growth. The y axis is the optical density of a 1/300 dilution of the culture. None, no antibiotic; Amp, 100 μg/ml ampicillin; Chlo, 34 μg/ml chloramphenicol; Kana, 50 μg/ml kanamycin; Tetra, 15 μg/ml tetracycline; Zeo, 100 μg/ml Zeocin. (B) Recombinant proteins purified by affinity chromatography on Ni-NTA beads from 4 ml culture. From 100 μl of elution volume, 15 μl were analyzed by SDS-PAGE along with 15 μl of non-purified supernatant (lanes S). M, molecular weight markers; from top to bottom: 116, 66.2, 45, 35, 25, 18.4, 14.4 kDa. Numbers 50, 100, 200 in lanes Chlo and Tetra indicate the volume in μl of 50% Ni-NTA beads suspension used. See results for details.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Antibiotics have no effect on P. pastoris growth nor on protein expression. (A) Effect of antibiotics on cell growth. The y axis is the optical density of a 1/300 dilution of the culture. None, no antibiotic; Amp, 100 μg/ml ampicillin; Chlo, 34 μg/ml chloramphenicol; Kana, 50 μg/ml kanamycin; Tetra, 15 μg/ml tetracycline; Zeo, 100 μg/ml Zeocin. (B) Recombinant proteins purified by affinity chromatography on Ni-NTA beads from 4 ml culture. From 100 μl of elution volume, 15 μl were analyzed by SDS-PAGE along with 15 μl of non-purified supernatant (lanes S). M, molecular weight markers; from top to bottom: 116, 66.2, 45, 35, 25, 18.4, 14.4 kDa. Numbers 50, 100, 200 in lanes Chlo and Tetra indicate the volume in μl of 50% Ni-NTA beads suspension used. See results for details.
Mentions: To check that antibiotics had really no effect on yeast growth, a clone of P. pastoris cells expressing GH5 from pGAPZαA was grown for 4 days in YPD in the presence or absence of the antibiotics listed above at concentrations used in laboratories to select resistant E. coli. Biomass was assessed by measuring the optical density of the culture at 600 nm at the end of that period, which definitely demonstrated the total innocuousness of this series of antibiotics (Figure 4A). The amount of recombinant protein secreted in the culture medium was next compared. To that end, His6-tagged GH5 was concentrated by affinity chromatography on Ni-NTA beads under different conditions that are detailed in the next paragraph. Results (Figure 4B) showed no difference between protein expression in the presence or absence of antibiotic, suggesting that antibiotics have no negative effect on the over expression of a recombinant protein in P. pastoris. In conclusion, if bacterial contaminations are an issue commonly used antibiotics can provide good protection at much lower cost than Zeocin, and without effect on P. pastoris cell growth or protein expression.

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