Expression of glycosylated haloalkane dehalogenase LinB in Pichia pastoris

Takashi Nakamura; Marcel Zámocký; Zbyněk Zdráhal; Radka Chaloupková; Marta Monincová; Zbyněk Prokop; Yuji Nagata; Jiří Damborský

doi:10.1016/j.pep.2005.08.022

Expression of glycosylated haloalkane dehalogenase LinB in Pichia pastoris

Takashi Nakamura, Marcel Zámocký, Zbyněk Zdráhal, Radka Chaloupková, Marta Monincová, Zbyněk Prokop, Yuji Nagata, Jiří Damborský

LIF - Molecular and Chemical Life Science

Research output: Contribution to journal › Article › peer-review

11 Citations (Scopus)

Abstract

Heterologous expression of the bacterial enzyme haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 in methylotrophic yeast Pichia pastoris is reported. The haloalkane dehalogenase gene linB was subcloned into the pPICZαA vector and integrated into the genome of P. pastoris. The recombinant LinB secreted from the yeast was purified to homogeneity and biochemically characterized. The deglycosylation experiment and mass spectrometry measurements showed that the recombinant LinB expressed in P. pastoris is glycosylated with a 2.8 kDa size of high mannose core. The specific activity of the glycosylated LinB was 15.6 ± 3.7 μmol/min/mg of protein with 1,2-dibromoethane and 1.86 ± 0.36 μmol/min/mg of protein with 1-chlorobutane. Activity and solution structure of the protein produced in P. pastoris is comparable with that of recombinant LinB expressed in Escherichia coli. The melting temperature determined by the circular dichroism (41.7 ± 0.3°C for LinB expressed in P. pastoris and 41.8 ± 0.3°C expressed in E. coli) and thermal stability measured by specific activity to 1-chlorobutane were also similar for two enzymes. Our results show that LinB can be extracellularly expressed in eukaryotic cell and glycosylation had no effect on activity, protein fold and thermal stability of LinB.

Original language	English
Pages (from-to)	85-91
Number of pages	7
Journal	Protein Expression and Purification
Volume	46
Issue number	1
DOIs	https://doi.org/10.1016/j.pep.2005.08.022
Publication status	Published - 2006 Mar

Keywords

Catalytic activity
Dehalogenation
Glycosylation
Heterologous expression
Pichia pastoris
Thermal stability

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10.1016/j.pep.2005.08.022

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@article{0315bbaf4aeb4bbb893561bb34569795,

title = "Expression of glycosylated haloalkane dehalogenase LinB in Pichia pastoris",

abstract = "Heterologous expression of the bacterial enzyme haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 in methylotrophic yeast Pichia pastoris is reported. The haloalkane dehalogenase gene linB was subcloned into the pPICZαA vector and integrated into the genome of P. pastoris. The recombinant LinB secreted from the yeast was purified to homogeneity and biochemically characterized. The deglycosylation experiment and mass spectrometry measurements showed that the recombinant LinB expressed in P. pastoris is glycosylated with a 2.8 kDa size of high mannose core. The specific activity of the glycosylated LinB was 15.6 ± 3.7 μmol/min/mg of protein with 1,2-dibromoethane and 1.86 ± 0.36 μmol/min/mg of protein with 1-chlorobutane. Activity and solution structure of the protein produced in P. pastoris is comparable with that of recombinant LinB expressed in Escherichia coli. The melting temperature determined by the circular dichroism (41.7 ± 0.3°C for LinB expressed in P. pastoris and 41.8 ± 0.3°C expressed in E. coli) and thermal stability measured by specific activity to 1-chlorobutane were also similar for two enzymes. Our results show that LinB can be extracellularly expressed in eukaryotic cell and glycosylation had no effect on activity, protein fold and thermal stability of LinB.",

keywords = "Catalytic activity, Dehalogenation, Glycosylation, Heterologous expression, Pichia pastoris, Thermal stability",

author = "Takashi Nakamura and Marcel Z{\'a}mock{\'y} and Zbyn{\v e}k Zdr{\'a}hal and Radka Chaloupkov{\'a} and Marta Monincov{\'a} and Zbyn{\v e}k Prokop and Yuji Nagata and Ji{\v r}{\'i} Damborsk{\'y}",

note = "Funding Information: This work was supported by postdoctoral fellowship for research abroad from Yamada Science Foundation (to T.N.). We thank Hana Kone{\v c}n{\'a} (Laboratory of Functional Genomics and Proteomics, Masaryk University, Brno, Czech Republic) for protein digestion for MS analysis. This work was supported by the grants from the Czech Ministry of Education (No. MSM 0021622412 and MSM0021622415). ",

year = "2006",

month = mar,

doi = "10.1016/j.pep.2005.08.022",

language = "English",

volume = "46",

pages = "85--91",

journal = "Protein Expression and Purification",

issn = "1046-5928",

publisher = "Academic Press Inc.",

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TY - JOUR

T1 - Expression of glycosylated haloalkane dehalogenase LinB in Pichia pastoris

AU - Nakamura, Takashi

AU - Zámocký, Marcel

AU - Zdráhal, Zbyněk

AU - Chaloupková, Radka

AU - Monincová, Marta

AU - Prokop, Zbyněk

AU - Nagata, Yuji

AU - Damborský, Jiří

N1 - Funding Information: This work was supported by postdoctoral fellowship for research abroad from Yamada Science Foundation (to T.N.). We thank Hana Konečná (Laboratory of Functional Genomics and Proteomics, Masaryk University, Brno, Czech Republic) for protein digestion for MS analysis. This work was supported by the grants from the Czech Ministry of Education (No. MSM 0021622412 and MSM0021622415).

PY - 2006/3

Y1 - 2006/3

N2 - Heterologous expression of the bacterial enzyme haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 in methylotrophic yeast Pichia pastoris is reported. The haloalkane dehalogenase gene linB was subcloned into the pPICZαA vector and integrated into the genome of P. pastoris. The recombinant LinB secreted from the yeast was purified to homogeneity and biochemically characterized. The deglycosylation experiment and mass spectrometry measurements showed that the recombinant LinB expressed in P. pastoris is glycosylated with a 2.8 kDa size of high mannose core. The specific activity of the glycosylated LinB was 15.6 ± 3.7 μmol/min/mg of protein with 1,2-dibromoethane and 1.86 ± 0.36 μmol/min/mg of protein with 1-chlorobutane. Activity and solution structure of the protein produced in P. pastoris is comparable with that of recombinant LinB expressed in Escherichia coli. The melting temperature determined by the circular dichroism (41.7 ± 0.3°C for LinB expressed in P. pastoris and 41.8 ± 0.3°C expressed in E. coli) and thermal stability measured by specific activity to 1-chlorobutane were also similar for two enzymes. Our results show that LinB can be extracellularly expressed in eukaryotic cell and glycosylation had no effect on activity, protein fold and thermal stability of LinB.

AB - Heterologous expression of the bacterial enzyme haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 in methylotrophic yeast Pichia pastoris is reported. The haloalkane dehalogenase gene linB was subcloned into the pPICZαA vector and integrated into the genome of P. pastoris. The recombinant LinB secreted from the yeast was purified to homogeneity and biochemically characterized. The deglycosylation experiment and mass spectrometry measurements showed that the recombinant LinB expressed in P. pastoris is glycosylated with a 2.8 kDa size of high mannose core. The specific activity of the glycosylated LinB was 15.6 ± 3.7 μmol/min/mg of protein with 1,2-dibromoethane and 1.86 ± 0.36 μmol/min/mg of protein with 1-chlorobutane. Activity and solution structure of the protein produced in P. pastoris is comparable with that of recombinant LinB expressed in Escherichia coli. The melting temperature determined by the circular dichroism (41.7 ± 0.3°C for LinB expressed in P. pastoris and 41.8 ± 0.3°C expressed in E. coli) and thermal stability measured by specific activity to 1-chlorobutane were also similar for two enzymes. Our results show that LinB can be extracellularly expressed in eukaryotic cell and glycosylation had no effect on activity, protein fold and thermal stability of LinB.

KW - Catalytic activity

KW - Dehalogenation

KW - Glycosylation

KW - Heterologous expression

KW - Pichia pastoris

KW - Thermal stability

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U2 - 10.1016/j.pep.2005.08.022

DO - 10.1016/j.pep.2005.08.022

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AN - SCOPUS:32644476853

SN - 1046-5928

VL - 46

SP - 85

EP - 91

JO - Protein Expression and Purification

JF - Protein Expression and Purification

IS - 1

ER -

Expression of glycosylated haloalkane dehalogenase LinB in Pichia pastoris

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Keywords

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