Overexpression in Escherichia coli and characterization of the chloroplast triosephosphate isomerase from spinach.
An important Calvin cycle enzyme, chloroplast triosephosphate isomerase (cpTPI) from spinach, has been cloned and expressed in up to 15% of the total cell protein using the P(L) expression vector in Escherichia coli. An even higher level expression, up to 36% of the total protein, was achieved by replacing the nucleotide sequence between the ribosomal binding site and the initial codon, ATG, with an AT-rich sequence. Computer modeling revealed that the moderate change in the standard free energy (5'-DeltaG degrees ) of mRNA secondary structure in the translation initial region might be the major factor which led to the later high-level expression. The overexpressed spinach cpTPI was soluble and fully active and was able to be purified beyond 95% purity by DEAE-Sepharose and Sephadex G-75, and around 55 mg of purified enzymes was obtained from 1 liter of cultured bacteria. With d-glyceraldehyde 3-phosphate as substrate, K(m (D-3-P)) is 0. 68 mM, V(max (G-3-P)) is 3.16 x 10(4) micromol/min. mg, and K(cat (G-3-P)) is 4.51 x 10(3)/s; with dihydroxyacetone phosphate as substrate, the corresponding values are 7.27 mM, 1.04 x 10(3) micromol/min. mg, and 1.16 x 10(2)/s, respectively.
Tang
G L
GL
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, 200032, China.
Wang
Y F
YF
Bao
J S
JS
Chen
H B
HB
D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018724:Spinacia oleracea / Q000201:enzymology*; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics*
D011994:Recombinant Proteins; D014305:Triose-Phosphate Isomerase