Identification and characterization of inosine monophosphate dehydrogenase from Halobacterium salinarum

Soo Jin Park, Won A. Joo, Jiyeon Choi, So Hee Lee, Chan Wha Kim

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Extremely halophilic Archaea, Halobacterium salinarum live in hypersaline habitats and maintain an osmotic balance of their cytoplasm by accumulating high concentrations of salt (mainly KCI). Therefore, their enzymes adapted to high NaCl concentrations offer a multitude of acutal or potential applications such as biocatalysts in the presence of high salt concentrations. In this study, the protein expression profile of H. salinarum cultured under different NaCl concentrations (3.5 M, 4.3 M, and 6.0 M) was investigated using two-dimensional gel electrophoresis (2-DE). As a result of 2-DE, the protein spots concentrated in acidic range at pH 3-10 were separated effectively using pH 3.5-4.5 ultrazoom IPG DryStrips. The proteins which proved to be upregulated or downregulated in 2-DE gel were digested with trypsin and identified with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization quadrupole (ESI-Q) TOF-mass spectrometry. Most proteins were identified as known annotated proteins based on sequence homology and few as unknown hypothetical proteins. Among proteins identified, an enzyme named inosine monophosphate dehydrogenase (IMPDH) was selected based on the possibility of its industrial application. IMPDH gene (1.6 kb fragment) expected to exist in H. salinarum was amplified by polymerase chain reaction (PCR) and expressed in Escherichia coli strain, BL21 (DE3) using a pGEX-KG vector. Recombinant IMPDH purified from H. salinarum has a higher activity in the presence of salt than in the absence of salt.

Original languageEnglish
Pages (from-to)3632-3641
Number of pages10
JournalProteomics
Volume4
Issue number11
DOIs
Publication statusPublished - 2004 Nov 1

Fingerprint

Halobacterium salinarum
Inosine Monophosphate
Oxidoreductases
Salts
Proteins
Enzymes
Gels
Electrospray ionization
Polymerase chain reaction
Archaea
Electrophoresis, Gel, Two-Dimensional
Sequence Homology
Electrophoresis
Trypsin
Escherichia coli
Industrial applications
Ionization
Mass spectrometry
Ecosystem
Desorption

Keywords

  • Electrospray ionization-quadrupole time of flight-mass spectrometry
  • Halobacterium salinarum
  • Inosine monophosphate dehydrogenase
  • Matrix-assisted laser desorption/ionization-time of flight
  • Two-dimensional gel electrophoresis

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Identification and characterization of inosine monophosphate dehydrogenase from Halobacterium salinarum. / Park, Soo Jin; Joo, Won A.; Choi, Jiyeon; Lee, So Hee; Kim, Chan Wha.

In: Proteomics, Vol. 4, No. 11, 01.11.2004, p. 3632-3641.

Research output: Contribution to journalArticle

Park, Soo Jin ; Joo, Won A. ; Choi, Jiyeon ; Lee, So Hee ; Kim, Chan Wha. / Identification and characterization of inosine monophosphate dehydrogenase from Halobacterium salinarum. In: Proteomics. 2004 ; Vol. 4, No. 11. pp. 3632-3641.
@article{ef251e455e624d239b2e1e30a26a3056,
title = "Identification and characterization of inosine monophosphate dehydrogenase from Halobacterium salinarum",
abstract = "Extremely halophilic Archaea, Halobacterium salinarum live in hypersaline habitats and maintain an osmotic balance of their cytoplasm by accumulating high concentrations of salt (mainly KCI). Therefore, their enzymes adapted to high NaCl concentrations offer a multitude of acutal or potential applications such as biocatalysts in the presence of high salt concentrations. In this study, the protein expression profile of H. salinarum cultured under different NaCl concentrations (3.5 M, 4.3 M, and 6.0 M) was investigated using two-dimensional gel electrophoresis (2-DE). As a result of 2-DE, the protein spots concentrated in acidic range at pH 3-10 were separated effectively using pH 3.5-4.5 ultrazoom IPG DryStrips. The proteins which proved to be upregulated or downregulated in 2-DE gel were digested with trypsin and identified with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization quadrupole (ESI-Q) TOF-mass spectrometry. Most proteins were identified as known annotated proteins based on sequence homology and few as unknown hypothetical proteins. Among proteins identified, an enzyme named inosine monophosphate dehydrogenase (IMPDH) was selected based on the possibility of its industrial application. IMPDH gene (1.6 kb fragment) expected to exist in H. salinarum was amplified by polymerase chain reaction (PCR) and expressed in Escherichia coli strain, BL21 (DE3) using a pGEX-KG vector. Recombinant IMPDH purified from H. salinarum has a higher activity in the presence of salt than in the absence of salt.",
keywords = "Electrospray ionization-quadrupole time of flight-mass spectrometry, Halobacterium salinarum, Inosine monophosphate dehydrogenase, Matrix-assisted laser desorption/ionization-time of flight, Two-dimensional gel electrophoresis",
author = "Park, {Soo Jin} and Joo, {Won A.} and Jiyeon Choi and Lee, {So Hee} and Kim, {Chan Wha}",
year = "2004",
month = "11",
day = "1",
doi = "10.1002/pmic.200400921",
language = "English",
volume = "4",
pages = "3632--3641",
journal = "Proteomics",
issn = "1615-9853",
publisher = "Wiley-VCH Verlag",
number = "11",

}

TY - JOUR

T1 - Identification and characterization of inosine monophosphate dehydrogenase from Halobacterium salinarum

AU - Park, Soo Jin

AU - Joo, Won A.

AU - Choi, Jiyeon

AU - Lee, So Hee

AU - Kim, Chan Wha

PY - 2004/11/1

Y1 - 2004/11/1

N2 - Extremely halophilic Archaea, Halobacterium salinarum live in hypersaline habitats and maintain an osmotic balance of their cytoplasm by accumulating high concentrations of salt (mainly KCI). Therefore, their enzymes adapted to high NaCl concentrations offer a multitude of acutal or potential applications such as biocatalysts in the presence of high salt concentrations. In this study, the protein expression profile of H. salinarum cultured under different NaCl concentrations (3.5 M, 4.3 M, and 6.0 M) was investigated using two-dimensional gel electrophoresis (2-DE). As a result of 2-DE, the protein spots concentrated in acidic range at pH 3-10 were separated effectively using pH 3.5-4.5 ultrazoom IPG DryStrips. The proteins which proved to be upregulated or downregulated in 2-DE gel were digested with trypsin and identified with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization quadrupole (ESI-Q) TOF-mass spectrometry. Most proteins were identified as known annotated proteins based on sequence homology and few as unknown hypothetical proteins. Among proteins identified, an enzyme named inosine monophosphate dehydrogenase (IMPDH) was selected based on the possibility of its industrial application. IMPDH gene (1.6 kb fragment) expected to exist in H. salinarum was amplified by polymerase chain reaction (PCR) and expressed in Escherichia coli strain, BL21 (DE3) using a pGEX-KG vector. Recombinant IMPDH purified from H. salinarum has a higher activity in the presence of salt than in the absence of salt.

AB - Extremely halophilic Archaea, Halobacterium salinarum live in hypersaline habitats and maintain an osmotic balance of their cytoplasm by accumulating high concentrations of salt (mainly KCI). Therefore, their enzymes adapted to high NaCl concentrations offer a multitude of acutal or potential applications such as biocatalysts in the presence of high salt concentrations. In this study, the protein expression profile of H. salinarum cultured under different NaCl concentrations (3.5 M, 4.3 M, and 6.0 M) was investigated using two-dimensional gel electrophoresis (2-DE). As a result of 2-DE, the protein spots concentrated in acidic range at pH 3-10 were separated effectively using pH 3.5-4.5 ultrazoom IPG DryStrips. The proteins which proved to be upregulated or downregulated in 2-DE gel were digested with trypsin and identified with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization quadrupole (ESI-Q) TOF-mass spectrometry. Most proteins were identified as known annotated proteins based on sequence homology and few as unknown hypothetical proteins. Among proteins identified, an enzyme named inosine monophosphate dehydrogenase (IMPDH) was selected based on the possibility of its industrial application. IMPDH gene (1.6 kb fragment) expected to exist in H. salinarum was amplified by polymerase chain reaction (PCR) and expressed in Escherichia coli strain, BL21 (DE3) using a pGEX-KG vector. Recombinant IMPDH purified from H. salinarum has a higher activity in the presence of salt than in the absence of salt.

KW - Electrospray ionization-quadrupole time of flight-mass spectrometry

KW - Halobacterium salinarum

KW - Inosine monophosphate dehydrogenase

KW - Matrix-assisted laser desorption/ionization-time of flight

KW - Two-dimensional gel electrophoresis

UR - http://www.scopus.com/inward/record.url?scp=8744225397&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=8744225397&partnerID=8YFLogxK

U2 - 10.1002/pmic.200400921

DO - 10.1002/pmic.200400921

M3 - Article

C2 - 15468334

AN - SCOPUS:8744225397

VL - 4

SP - 3632

EP - 3641

JO - Proteomics

JF - Proteomics

SN - 1615-9853

IS - 11

ER -