Cloning and High Expression in E.coli of a Chimeric Gene Coding for Rice Fructose-l,6-bisphosphate Aldolase.
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.
Chen
Hai-Bao
HB
State 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.
Yang
Chun-Song
CS