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High-level expression of the native barley α-amylase/subtilisin inhibitor in Pichia pastoris

Paper ID Volume ID Publish Year Pages File Format Full-Text
24962 43549 2008 9 PDF Available
Title
High-level expression of the native barley α-amylase/subtilisin inhibitor in Pichia pastoris
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.

Keywords
BASI, barley α-amylase/subtilisin inhibitor; AOX1, alcohol oxidase 1; PCR, polymerase chain reaction; AMY2, α-amylase 2; WASI, wheat α-amylase/subtilisin inhibitor; RASI, rice α-amylase/subtilisin inhibitor; CDR, coding regionBarley α-amylase/subtilisin i
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High-level expression of the native barley α-amylase/subtilisin inhibitor in Pichia pastoris
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 133, Issue 4, 29 February 2008, Pages 424–432
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us