title: Cloning and expression of hemicellulases from Aspergillus nidulans in Pichia pastoris.
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.
authors.affiliation: Food Safety and Analytical Quality Department, Central Food Technological Research Institute (CSIR), Cheluvamba Mansion, Mysore, India. pvasu@astate.edu
authors.forename: Prasanna
authors.initials: P
authors.lastname: Vasu
authors.forename: Stefan
authors.initials: S
authors.lastname: Bauer
authors.forename: Brett J
authors.initials: BJ
authors.lastname: Savary
mesh_terms: D001233:Aspergillus nidulans / Q000201:enzymology*; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D015151:Immunoblotting; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C023305:hemicellulase