TY - JOUR
T1 - A new strategy for the cloning, overexpression and one step purification of three DHAP-dependent aldolases
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
N1 - 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).
PY - 1995/7
Y1 - 1995/7
N2 - 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.
AB - 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.
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U2 - 10.1016/0968-0896(95)00077-T
DO - 10.1016/0968-0896(95)00077-T
M3 - Article
C2 - 7582972
AN - SCOPUS:0029055668
VL - 3
SP - 945
EP - 953
JO - Bioorganic and Medicinal Chemistry
JF - Bioorganic and Medicinal Chemistry
SN - 0968-0896
IS - 7
ER -