A new strategy for the cloning, overexpression and one step purification of three DHAP-dependent aldolases: Rhamnulose-1-phosphate aldolase, fuculose-1-phosphate aldolase and tagatose-1,6-diphosphate aldolase1

Eduardo Garcia-Junceda, Gwo Jenn Shen, Takeshi Sugai, Chi Huey Wong

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Three DHAP-dependent aldolases, rhamnulose-1-phosphate aldolase (Rham-IPA), fuculose-1-phosphate aldolase (Fuc-1PA) and tagatose-1,6-diphosphate aldolase (TDPA) have been cloned and overexpressed in Escherichia coli using two different expression vectors: pTrcHis for the expression of Rham-1PA and Fuc-1PA and pRSET for the expression of TDPA. In each case the recombinant enzyme is synthesized as a fusion protein with a hexahistidine tag on the N-terminus. The three enzymes have been purified in only one step by chelation affinity chromatography. The effects of cultivation temperature and concentration of inducer have been studied in order to optimize the expression of the recombinant proteins and to avoid the formation of inclusion bodies.

Original languageEnglish
Pages (from-to)945-953
Number of pages9
JournalBioorganic and Medicinal Chemistry
Volume3
Issue number7
DOIs
Publication statusPublished - 1995
Externally publishedYes

Fingerprint

Aldehyde-Lyases
Fructose-Bisphosphate Aldolase
His-His-His-His-His-His
Cloning
Purification
Organism Cloning
Affinity chromatography
Enzymes
Chelation
Recombinant Proteins
Escherichia coli
Fusion reactions
Inclusion Bodies
Affinity Chromatography
Proteins
Temperature
rhamnulose 1-phosphate
tagatose 1,6-diphosphate aldolase
fuculose 1-phosphate

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Molecular Biology
  • Molecular Medicine
  • Organic Chemistry
  • Drug Discovery
  • Pharmaceutical Science

Cite this

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title = "A new strategy for the cloning, overexpression and one step purification of three DHAP-dependent aldolases: Rhamnulose-1-phosphate aldolase, fuculose-1-phosphate aldolase and tagatose-1,6-diphosphate aldolase1",
abstract = "Three DHAP-dependent aldolases, rhamnulose-1-phosphate aldolase (Rham-IPA), fuculose-1-phosphate aldolase (Fuc-1PA) and tagatose-1,6-diphosphate aldolase (TDPA) have been cloned and overexpressed in Escherichia coli using two different expression vectors: pTrcHis for the expression of Rham-1PA and Fuc-1PA and pRSET for the expression of TDPA. In each case the recombinant enzyme is synthesized as a fusion protein with a hexahistidine tag on the N-terminus. The three enzymes have been purified in only one step by chelation affinity chromatography. The effects of cultivation temperature and concentration of inducer have been studied in order to optimize the expression of the recombinant proteins and to avoid the formation of inclusion bodies.",
author = "Eduardo Garcia-Junceda and Shen, {Gwo Jenn} and Takeshi Sugai and Wong, {Chi Huey}",
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T2 - Rhamnulose-1-phosphate aldolase, fuculose-1-phosphate aldolase and tagatose-1,6-diphosphate aldolase1

AU - Garcia-Junceda, Eduardo

AU - Shen, Gwo Jenn

AU - Sugai, Takeshi

AU - Wong, Chi Huey

PY - 1995

Y1 - 1995

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