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High-level expression of the native barley alpha-amylase/subtilisin inhibitor in Pichia pastoris
- Source :
- Journal of biotechnology. 133(4)
- Publication Year :
- 2007
-
Abstract
- An expression system for high-level expression of the native Hordeum vulgare α-amylase/subtilisin inhibitor (BASI) has been developed in Pichia pastoris , using the methanol inducible alcohol oxidase 1 (AOX1) promoter. To optimize expression, two codon-optimized coding regions have been designed and expressed alongside the wild-type coding region. To ensure secretion of the native mature protein, a truncated version of the alpha mating factor secretion signal from Saccharomyces cerevisiae was used. In order to be able to compare expression levels from different clones, single insertion transformants generated by gene replacement of the AOX1 gene was selected by PCR screening. Following methanol induction, expression levels reached 125 mg L −1 from the wild-type coding region while expression from the two codon-optimized variants reached 65 and 125 mg L −1 , respectively. The protein was purified and characterized by Edman degradation, liquid chromatography mass spectrometry and insoluble blue starch assay, and was shown to posses the same characteristics as wild-type protein purified from barley grains.
- Subjects :
- Molecular Sequence Data
Gene Expression
Bioengineering
Applied Microbiology and Biotechnology
Polymerase Chain Reaction
Pichia
Pichia pastoris
Gene expression
Amylase
Amino Acid Sequence
Gene
Plant Proteins
biology
Edman degradation
Base Sequence
Models, Genetic
Sequence Homology, Amino Acid
Subtilisin
General Medicine
biology.organism_classification
Molecular biology
Recombinant Proteins
Blotting, Southern
Biochemistry
biology.protein
Electrophoresis, Polyacrylamide Gel
Hordeum vulgare
Heterologous expression
Trypsin Inhibitor, Kunitz Soybean
Sequence Alignment
Biotechnology
Subjects
Details
- ISSN :
- 01681656
- Volume :
- 133
- Issue :
- 4
- Database :
- OpenAIRE
- Journal :
- Journal of biotechnology
- Accession number :
- edsair.doi.dedup.....319ab334b15626c3ad94cae0e13c8ddd