Overexpression in Escherichia coli and characterization of the chloroplasttriosephosphate isomerase from spinach

An important Calvin cycle enzyme, chloroplast triosephosphate isomerase (cp TPI) 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 h igher level expression, up to 36% of the total protein, was achieved by rep lacing the nucleotide sequence between the ribosomal binding site and the i nitial codon, ATG, with an AT-rich sequence. Computer modeling revealed tha t the moderate change in the standard free energy (5'-Delta G degrees) of m RNA secondary structure in the translation initial region might be the majo r factor which led to the later high-level expression. The overexpressed sp inach 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 purifi ed enzymes was obtained from 1 liter of cultured bacteria. With D-glycerald ehyde 3-phosphate as substrate, Km (D-3-P) is 0.68 mM, Vmax (G-3-P) is 3.16 x 10(4) mu mol/ min . mg, and Kcat (G-3-P) is 4.51 x 10(3)/s; with dihydro xyacetone phosphate as substrate, the corresponding values are 7.27 mM, 1.0 4 x 10(3) mu mol/min . mg, and 1.16 x 10(2)/s, respectively. (C) 1999 Acade mic Press.