title: Overexpression, purification, and characterization of recombinant barley alpha-amylases 1 and 2 secreted by the methylotrophic yeast Pichia pastoris.
abstract: Recombinant barley alpha-amylase isozymes 1 and 2 were secreted by Pichia pastoris at up to 50 and 1 mg/liter, respectively, representing approximately a 50-fold increase compared to the levels of the heterologous expression by Saccharomyces cerevisiae. The cDNA clones E or pM/C encoding isozymes 1 and 2, respectively, were placed under the control of regulatory sequences from the Pichia AOX1 gene in the vector pHIL-D2. Both isozymes were effectively secreted to the medium as directed by their own signal sequences and easily purified to homogeneity in quantitative yield by affinity chromatography on beta-cyclodextrin-Sepharose. The N-terminal sequence, pI, and Mr indicated that native-like processing took place. Electrospray ionization mass spectrometry, however, revealed microheterogeneity for recombinant isozyme 1. While Mr of one recombinant isozyme 1 form of 45,452 was in excellent agreement with a value of 45,447 calculated from the sequence, liquid chromatography/mass spectrometry of endo Lys C-generated peptides followed by tandem mass spectrometry on a nanoelectrospray ionization/mass spectrometry/mass spectrometry system identified additional recombinant isozyme 1 forms to be glycosylated on Thr410, N-acetylated on His1, S-glutathionylated on Cys95, or C-terminally truncated of -412RS, -411QRS, and -410LQRS. The recombinant enzymes and the alpha-amylases from barley malt closely resembled each other in enzymatic activity on insoluble Blue Starch, amylose of degree of polymerization 17, and 2-chloro-4-nitrophenyl beta-D-maltoheptaoside as well as in Ca2+ dependency of activity. Pichia pastoris thus produced in high yields recombinant alpha-amylase that is similar with respect to structure and function to the enzyme purified from malt extracts. This greatly facilitates future mutational analysis of barley alpha-amylase in order to probe structure/function relationships.
authors.affiliation: Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, Copenhagen Valby, DK-2500, Denmark.
authors.forename: N
authors.initials: N
authors.lastname: Juge
authors.forename: J S
authors.initials: JS
authors.lastname: Andersen
authors.forename: D
authors.initials: D
authors.lastname: Tull
authors.forename: P
authors.initials: P
authors.lastname: Roepstorff
authors.forename: B
authors.initials: B
authors.lastname: Svensson
mesh_terms: D000681:Amylases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002845:Chromatography; D003001:Cloning, Molecular; D017931:DNA Primers; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D001467:Hordeum / Q000201:enzymology*; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013213:Starch / Q000378:metabolism; D014170:Transformation, Genetic / Q000235:genetics
chemical_list: D017931:DNA Primers; D010940:Plant Proteins; D011994:Recombinant Proteins; D013213:Starch; D000681:Amylases