Overproduction, in Escherichia coli, of soluble taxadiene synthase, a key enzyme in the Taxol biosynthetic pathway.
Taxadiene synthase catalyzes the conversion of the universal precursor of diterpenoids, geranylgeranyl diphosphate, to taxadiene, a key intermediate in Taxol (paclitaxel) biosynthesis. The gene encoding taxadiene synthase was cloned recently. Here we report a method for the heterologous overexpression of cDNA encoding taxadiene synthase in Escherichia coli using a thioredoxin fusion expression system, which increases the solubility of expressed protein. Taxadiene synthase cDNA was amplified by polymerase chain reaction and then subcloned into pET3d and pET32a(+) to form pET3dTX and pET32TX, respectively. The expressed taxadiene synthase from E. coli BL21(DE3)/pET3dTX was present completely as inclusion bodies. The transformant E. coli BL21(DE3)/pET32TX produced a thioredoxin fusion taxadiene synthase (15-20% of total soluble protein) when induced with isopropyl beta-D-thiogalactopyranoside at low temperature (20 degrees C). The recombinant enzyme was purified by a single step with a His-binding metal affinity column. The maximal production attained was 13 mg of purified, active fusion protein per 500 ml culture of E. coli BL21(DE3)/pET32TX. The purified recombinant taxadiene synthase fusion protein was similar to native protein in steady-state kinetic parameters and mobility on sodium sulfate-polyacrylamide gel electrophoresis. The protein purified from E. coli BL21(DE3)/pET3dTX had the expected N-terminal (AQLSFNA) sequence.
Huang
K X
KX
Chemistry Department, Texas A&M University, College Station 77843-3255, USA.
Huang
Q L
QL
Wildung
M R
MR
Croteau
R
R
Scott
A I
AI
D000972:Antineoplastic Agents, Phytogenic / Q000096:biosynthesis*; D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D007535:Isomerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017239:Paclitaxel / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D000972:Antineoplastic Agents, Phytogenic; D017931:DNA Primers; D011994:Recombinant Proteins; D007535:Isomerases; C095926:taxa-4(5),11(12)-diene synthase; D017239:Paclitaxel