title: Overexpression in Escherichia coli and characterization of the chloroplast triosephosphate isomerase from spinach.
abstract: 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.
authors.affiliation: Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, 200032, China.
authors.forename: G L
authors.initials: GL
authors.lastname: Tang
authors.forename: Y F
authors.initials: YF
authors.lastname: Wang
authors.forename: J S
authors.initials: JS
authors.lastname: Bao
authors.forename: H B
authors.initials: HB
authors.lastname: Chen
mesh_terms: 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*
chemical_list: D011994:Recombinant Proteins; D014305:Triose-Phosphate Isomerase