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Cloning and High Expression in E.coli of a Chimeric Gene Coding for Rice Fructose-l,6-bisphosphate Aldolase
1State Key Laboratory of Bioorganic and Natural Products Chemistry, the Laboratory of Computer Chemistry, Shanghai Institute of Organic Chemistry, the Chinese Academy of Sciences, Shanghai 200032, China.
Abstract:
A chimeric gene (l 104 bp) coding for rice fructose-l,6-bisphosphate aldolase has been constructed by DNA recombination of a synthetic 5'- fragment (-24 to 272) and an RT-PCR amplified product at restriction site S fu I. The synthetic fragment was assembled from six oligonucleotides by T4 DNA ligase reaction according to a single-stranded DNA method (Chen H-B et al, Nucleic Acids Res 1990, 18, 871-878), the PCR amplified fragment (217 - 1 080) was obtained by carrying out a PCR in the presence of rice cDNA as the template and both the 5'- and the 3'- primers. The whole gene was successively cloned into plasmids pWR13 and pPLc2833, and highly expressed in E. coli to produce the expected product. After purification through stepwise precipitation and cation-exchange column chromatography, the recombinant aldolase showed an enzyme activity as high as (1l.0 +/- 0.3) units/mg with the turnover number = 27s(-1) and K(m) (FBP)=4.2 &mgr;M by two different methods.