Details
| Original language | English |
|---|---|
| Pages (from-to) | 233-239 |
| Number of pages | 7 |
| Journal | Protein Expression and Purification |
| Volume | 95 |
| Publication status | Published - 15 Jan 2014 |
Abstract
The first heterologous expression of an iron-containing lipoxygenase from a basidiomycete in Pichia pastoris is reported. Five different expression constructs of the lipoxygenase gene LOX1 from Pleurotus sapidus were cloned and successfully transferred into P. pastoris SMD1168, but only one pPIC9K vector construct was functionally expressed. In this construct the vector-provided α-factor signal sequence was replaced by insertion of a second Kozak sequence between the signal sequence and the LOX1 gene. His+ transformants were screened for their level of resistance to geneticin (G418). Lox1 was expressed under different culture conditions and purified using the N-terminal His-tag. Relative enzyme activity increased significantly 48 h after methanol induction and was highest with 2 ml l-1 inducer. The recombinant enzyme showed an optimal lipoxygenase activity at pH 7 and 30-35 C and a vmax like the wild-type enzyme.
Keywords
- Functional expression, Kozak sequence, Lipoxygenase, Pichia pastoris, Pleurotus sapidus
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
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In: Protein Expression and Purification, Vol. 95, 15.01.2014, p. 233-239.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Expression of soluble recombinant lipoxygenase from Pleurotus sapidus in Pichia pastoris
AU - Kelle, Sebastian
AU - Zelena, Katerina
AU - Krings, Ulrich
AU - Linke, Diana
AU - Berger, Ralf G.
N1 - Funding information: Support of the work by the BMBF cluster Biokatalyse2021 ( FKZ0315172B ) is gratefully acknowledged.
PY - 2014/1/15
Y1 - 2014/1/15
N2 - The first heterologous expression of an iron-containing lipoxygenase from a basidiomycete in Pichia pastoris is reported. Five different expression constructs of the lipoxygenase gene LOX1 from Pleurotus sapidus were cloned and successfully transferred into P. pastoris SMD1168, but only one pPIC9K vector construct was functionally expressed. In this construct the vector-provided α-factor signal sequence was replaced by insertion of a second Kozak sequence between the signal sequence and the LOX1 gene. His+ transformants were screened for their level of resistance to geneticin (G418). Lox1 was expressed under different culture conditions and purified using the N-terminal His-tag. Relative enzyme activity increased significantly 48 h after methanol induction and was highest with 2 ml l-1 inducer. The recombinant enzyme showed an optimal lipoxygenase activity at pH 7 and 30-35 C and a vmax like the wild-type enzyme.
AB - The first heterologous expression of an iron-containing lipoxygenase from a basidiomycete in Pichia pastoris is reported. Five different expression constructs of the lipoxygenase gene LOX1 from Pleurotus sapidus were cloned and successfully transferred into P. pastoris SMD1168, but only one pPIC9K vector construct was functionally expressed. In this construct the vector-provided α-factor signal sequence was replaced by insertion of a second Kozak sequence between the signal sequence and the LOX1 gene. His+ transformants were screened for their level of resistance to geneticin (G418). Lox1 was expressed under different culture conditions and purified using the N-terminal His-tag. Relative enzyme activity increased significantly 48 h after methanol induction and was highest with 2 ml l-1 inducer. The recombinant enzyme showed an optimal lipoxygenase activity at pH 7 and 30-35 C and a vmax like the wild-type enzyme.
KW - Functional expression
KW - Kozak sequence
KW - Lipoxygenase
KW - Pichia pastoris
KW - Pleurotus sapidus
UR - http://www.scopus.com/inward/record.url?scp=84893516091&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2014.01.004
DO - 10.1016/j.pep.2014.01.004
M3 - Article
C2 - 24440506
AN - SCOPUS:84893516091
VL - 95
SP - 233
EP - 239
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
ER -