Expression of soluble recombinant lipoxygenase from Pleurotus sapidus in Pichia pastoris

Research output: Contribution to journalArticleResearchpeer review

Authors

  • Sebastian Kelle
  • Katerina Zelena
  • Ulrich Krings
  • Diana Linke
  • Ralf G. Berger

Research Organisations

View graph of relations

Details

Original languageEnglish
Pages (from-to)233-239
Number of pages7
JournalProtein Expression and Purification
Volume95
Publication statusPublished - 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

Cite this

Expression of soluble recombinant lipoxygenase from Pleurotus sapidus in Pichia pastoris. / Kelle, Sebastian; Zelena, Katerina; Krings, Ulrich et al.
In: Protein Expression and Purification, Vol. 95, 15.01.2014, p. 233-239.

Research output: Contribution to journalArticleResearchpeer review

Kelle S, Zelena K, Krings U, Linke D, Berger RG. Expression of soluble recombinant lipoxygenase from Pleurotus sapidus in Pichia pastoris. Protein Expression and Purification. 2014 Jan 15;95:233-239. doi: 10.1016/j.pep.2014.01.004
Kelle, Sebastian ; Zelena, Katerina ; Krings, Ulrich et al. / Expression of soluble recombinant lipoxygenase from Pleurotus sapidus in Pichia pastoris. In: Protein Expression and Purification. 2014 ; Vol. 95. pp. 233-239.
Download
@article{a50286fe4e074172b81bc5d38035f926,
title = "Expression of soluble recombinant lipoxygenase from Pleurotus sapidus in Pichia pastoris",
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",
author = "Sebastian Kelle and Katerina Zelena and Ulrich Krings and Diana Linke and Berger, {Ralf G.}",
note = "Funding information: Support of the work by the BMBF cluster Biokatalyse2021 ( FKZ0315172B ) is gratefully acknowledged.",
year = "2014",
month = jan,
day = "15",
doi = "10.1016/j.pep.2014.01.004",
language = "English",
volume = "95",
pages = "233--239",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",

}

Download

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 -