Expression of Giardia duodenalis beta-tubulin as a soluble protein in Escherichia coli.
The beta-tubulin gene of the parasitic protozoan Giardia duodenalis has been expressed for the first time using a novel and direct method. The protein was expressed in both soluble and insoluble forms in an Escherichia coli-based expression system. The level of expression was found to be affected by several variables including the incubation temperature, length of time for which expression was carried out, and the E. coli culture volume. The protein expression system contributed no additional amino acids to the final fusion protein and the polyhistidine fusion sequence was easily removed from the beta-tubulin protein using a specific enterokinase enzyme. The expression system also provided a means of preparing a soluble protein and purifying it by a relatively straightforward affinity chromatography method to give a very high level of protein purity. This makes the protein suitable for a number of applications for characterization including beta-tubulin antibody assays, alpha-/beta-tubulin-binding regions, and beta-tubulin folding intermediates.
MacDonald
L M
LM
Division of Veterinary and Biomedical Sciences, Murdoch University, South Street, Murdoch, Western Australia, 6150. lmacdona@central.murdoch.edu.au
Armson
A
A
Thompson
R C
RC
Reynoldson
J A
JA
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004765:Enteropeptidase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015870:Gene Expression; D005872:Giardia / Q000235:genetics*; D006639:Histidine; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013696:Temperature; D013997:Time Factors; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D010455:Peptides; D011993:Recombinant Fusion Proteins; D014404:Tubulin; C033223:polyhistidine; D006639:Histidine; D004765:Enteropeptidase