| The diterpene Taxol is an important drug for the treatment of a variety of cancers. The low yield of Taxol from natural sources and increasing applications of Taxol in chemotherapy have prompted intense efforts to improve biological production yield of the drug, that depends critically upon a detailed understanding of the biosynthetic pathways leading to Taxol and related taxoids and definition of the responsible enzymes and genes.The biosynthesis of Taxol from plant primary metabolism is a very complex process, including at least 20 steps to construct its tetracyclic skeleton and the addition of various hydroxy and acyl functional groups. Many results from Taxol biogenetic schemes show that hydroxylations and acylations are important for the bioactivity of Taxol.As a part of work on gene cloning of Taxol biosynthesis, a new full-length acyltransferase gene was obtained by homology-based PCR cloning strategy. The cDNA has an open-reading frame of 1,275 nucleotides, which encodes 425 amino acids with a calculated molecular weight of 47,241 Da and an estimated pi of 5.93. The deduced amino acid sequence resembles the sequences of other cloned acyltransferases (56-61% identity; 71-75% similarity) involved directly in taxol biosynthetic pathways. More soluble protein could be produced when this cDNA was expressed in Escherichia coli using expression fusion vector than using non-fusion vectors. Through further trial-and-error experiments, a set of culture conditions that allowed production of a reasonable amount of soluble enzyme was established. SDS-PAGE result showed that a protein with an apparent molecular mass of 65kDa appeared, supporting that the new acyltransferase gene clone could be normally translated into a complete trxA-fusion protein as expected. The trxA-fusion protein was purified via nickel-affinity chromatography using HiTrap Chelating HP column. Expressed fusion-protein would be used for further enzymatic characterization.Because of all Taxus species are known to be very closely related only minor... |
