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

doi:10.1016/0968-0896(95)00077-T

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 journal › Article › peer-review

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 language	English
Pages (from-to)	945-953
Number of pages	9
Journal	Bioorganic and Medicinal Chemistry
Volume	3
Issue number	7
DOIs	https://doi.org/10.1016/0968-0896(95)00077-T
Publication status	Published - 1995 Jul
Externally published	Yes

ASJC Scopus subject areas

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

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Garcia-Junceda, E., Shen, G. J., Sugai, T., & Wong, C. H. (1995). 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. Bioorganic and Medicinal Chemistry, 3(7), 945-953. https://doi.org/10.1016/0968-0896(95)00077-T

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

In: Bioorganic and Medicinal Chemistry, Vol. 3, No. 7, 07.1995, p. 945-953.

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

Garcia-Junceda, E, Shen, GJ, Sugai, T & Wong, CH 1995, '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', Bioorganic and Medicinal Chemistry, vol. 3, no. 7, pp. 945-953. https://doi.org/10.1016/0968-0896(95)00077-T

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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}",

note = "Funding Information: We thank Professor Juan Aguilar for providing us the sequence of the gen rhaD before its publication. Also we thank Dr Ramtn Alajarin, Ms Mui-Mui Sire and Mr Wen-Chih Chou for the synthesis of the rhanmulose-1-phosphate, fuculose-l-phosphate and tagatose-l,6-diphosphate respectively. E. G.-J. was supported by a fellowship from C.S.I.C. (Spain). This work was supported by the NIH (GM 44145).",

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