TY - JOUR
T1 - High-level expression of the native barley α-amylase/subtilisin inhibitor in Pichia pastoris
AU - Ollendorff Micheelsen, Pernille
AU - Rahbek Østergaard, Peter
AU - Lange, Lene
AU - Skjøt, Michael
N1 - Keywords: Barley -amylase/subtilisin inhibitor (BASI); Pichia pastoris; Codon optimization; Heterologous expression; Truncated alpha mating factor secretion signal
PY - 2008
Y1 - 2008
N2 - 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.
AB - 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.
U2 - 10.1016/j.jbiotec.2007.11.012
DO - 10.1016/j.jbiotec.2007.11.012
M3 - Journal article
C2 - 18207271
SN - 0168-1656
VL - 133
SP - 424
EP - 432
JO - Journal of Biotechnology
JF - Journal of Biotechnology
IS - 4
ER -