Abstract
The methylotrophic yeast Pichia pastoris is increasingly used for heterologous expression of high quality proteins in laboratory-scale (milligram) quantities. Commercially available polysaccharide-active enzyme preparations have limited applications in plant cell wall research due to their heterogeneous mix of hydrolytic activities. P. pastoris provides an ideal in vitro expression system for producing monocomponent enzymes, since it lacks endogenous plant cell wall-active enzymes and can perform eukaryotic post-translational modifications (i.e., glycosylation). We have routinely prepared cDNA constructs from Aspergillus nidulans encoding a broad array of hydrolases active on various linkages contained in plant cell wall polysaccharides. The cDNAs were inserted into the pPICZα C shuttle vector (Invitrogen) in-frame with the Saccharomyces cerevisiae α-secretion factor and expressed under the transcriptional control of the highly inducible alcohol oxidase 1 (AOX1) promoter. The enzyme products were efficiently secreted into buffered complex methanol medium (BMMY) as C-terminal his-tagged proteins for simple one-step affinity purification. The insertion of the c-Myc epitope enabled easy immunodetection. Here we present the detailed protocols for primer design, cloning, expression, and activity assays for a representative set of xylan-acting hemicellulases produced in P. pastoris.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Higgins, D. R. and Cregg, J. M. (1998). Introduction to Pichia pastoris, in Methods in Molecular Biology: Pichia Protocols (Higgins, D. R. and Cregg, J. M., eds.), Humana, Totowa, NJ, Vol. 103, pp. 1–15.
Cereghino, J. L. and Cregg, J. M. (2000). Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev. 24, 45–66.
Cregg, J. M., Vedvick, T. S., and Raschke, W. C. (1993). Recent advances in expression of foreign genes in Pichia pastoris. BioTechnol. 11, 905–910.
Schutter, K. D., Lin, Y-C., Tiels, P., Hecke, A. V., Glinka, S., Weber-Lehmann, J., Rouze, P., Peer, Y. V., and Callewaert, N. (2009). Genome sequence of the recombinant protein production host Pichia pastoris. Nature Biotechnol. 27, 561–566.
Mort, A. J., Gao, S., Wu, X., and Prade, R. (2005). The need for pure enzymes that degrade cell wall polysaccharides and how to get them. Plant Biosystems 139, 84–87.
Galagan, J. E., Calvo, S. E., Ma, L-J., Wortman, J. R., Batzoglou, S., Lee, S-I., et al., (2005). Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature 438, 1105–1115.
Aspergillus Sequencing Project. Broad Institute of MIT and Harvard (http://www.broad.mit.edu).
Bauer, S., Vasu, P., Persson, S., Mort, A. J., and Somerville, C. R. (2006). Development and application of a suite of polysaccharide-degrading enzymes for analyzing plant cell walls. Proc. Natl. Acad. Sci. USA 103, 11417–11422.
Bauer, S., Vasu, P., Mort, A. J., and Somerville, C. R. (2005). Cloning, expression, and characterization of an oligoxyloglucan reducing end-specific xyloglucanobiohydrolase from Aspergillus nidulans. Carbohydr. Res. 340, 2590–2597.
Vasu, P., Bauer, S., Somerville, C. R., and Mort, A. J. (2011). Characterization and substrate specificity of recombinant xylanolytic enzymes: Potential analytical tools for the study of plant cell wall xylans. (Revised for Biochem. J).
Sambrook, J. and Russell, D. W. (eds.) (2001). Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Cregg, J. M. and Russell, K. A (1998). Transformation, in Methods in Molecular Biology: Pichia Protocols (Higgins, D. R. and Cregg, J. M., eds.), Humana, Totowa, NJ, Vol. 103, pp. 27–39.
Invitrogen (2009). EasySelect™ Pichia Expression Kit – For expression of recombinant proteins using pPICZ and pPICZα in Pichia pastoris, User Manual, Version I (Cat. No. K1740-0).
Invitrogen (2008). pPICZ A, B, and C – Pichia expression vectors for selection on Zeocin™ and purification of recombinant proteins, User Manual, Version E (Cat. No. V190-20).
Invitrogen (2003). NuPAGE Technical Guide – General information and protocols for using the NuPAGE electrophoresis system. Instruction Manual, Version E (Cat. No. IM-1001).
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.
Acknowledgments
The authors thank Drs. Andrew J. Mort (Oklahoma State University) and Christopher R. Somerville (University of California, Berkeley) for their support and supervision of this study. This was funded by the U.S. Department of Energy, Basic Energy Sciences Division grant DE-FG02-03ER15444 (to A.J.M. and C.R.S.) and USDOE Office of Energy Efficiency and Renewable Energy, Mid-South/Southeast Biofuels Consortium, DE-FG36-08BO88036 (to B.J.S.) Postdoctoral fellowships for S.B. provided by the Josef-Schormüller-Gedächtnisstiftung and the Deutsche Forschungsgemeinschaft.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Vasu, P., Bauer, S., Savary, B.J. (2012). Cloning and Expression of Hemicellulases from Aspergillus nidulans in Pichia pastoris . In: Lorence, A. (eds) Recombinant Gene Expression. Methods in Molecular Biology, vol 824. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-433-9_21
Download citation
DOI: https://doi.org/10.1007/978-1-61779-433-9_21
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-432-2
Online ISBN: 978-1-61779-433-9
eBook Packages: Springer Protocols