title: Expression cloned cDNA for 10-deacetylbaccatin III-10-O-acetyltransferase in Escherichia coli: a comparative study of three fusion systems.
abstract: 10-Deacetylbaccatin III-10-O-acetyltransferase (10-DABT) catalyzes the formation of baccatin III, which is an immediate diterpenoid precursor of Taxol. A cDNA encoding 10-DABT was cloned from Taxus baccata by using RT-PCR and screening a cDNA library. A study of its heterologous overexpression in Escherichia coli was carried out. To get high-level expression of recombinant enzyme, three kinds of IPTG inducible fusion expression systems (with glutathione S-transferase (GST), hexahistidine (6x His), and biotinylated tag) were used, and results of expression were compared. Fusion 10-DABT with different tags was expressed with diverse expression levels and solubility in the three systems. Optimum IPTG concentration, temperature, and inducing time for producing recombinant enzymes were found. Under higher IPTG concentration (up to 1 mM), the highest level of expression for fusion protein was obtained in the 6x His fusion system with phage T5 promoter, but expressed products were only partially soluble. With lower IPTG concentration (less than 0.5 mM), the highest expression was detected in the GST fusion system with tac promoter, and the lowest level of expression appeared in the biotinylated fusion system. The expression level in the latter system did not differ dramatically with a range of different inducer concentrations. GST and 6x His fusion proteins were mainly soluble in aqueous solutions and Triton X-100 improved the solubility of biotinylated fusion proteins (inferring this protein is membrane-associated). Fusion proteins could only be partially purified by a single affinity chromatography step for all three systems. Glutathione-coupled matrix and streptavidin-conjugated resin have higher specificity than Ni-NTA resin, and elution conditions were shown to affect enzyme activity. Three kinds of recombinant 10-DABT with different tags showed enzyme activity, but total enzyme activity was lost as a result of the affinity chromatography step. Thrombin and Factor Xa could be used for site-specific cleavage of fusion proteins, but the incubation temperature affected enzyme activity of recombinant enzymes.
authors.affiliation: School of Biology, University of Leeds, Leeds LS2 9JT, UK. bgyjfang@leeds.ac.uk
authors.forename: Jianjun
authors.initials: J
authors.lastname: Fang
authors.forename: Dietrich
authors.initials: D
authors.lastname: Ewald
mesh_terms: D000123:Acetyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D043823:Taxoids / Q000378:metabolism*; D013696:Temperature
chemical_list: D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D043823:Taxoids; C042213:10-deacetylbaccatine III; D000123:Acetyltransferases