Expression of the alpha and beta tubulin genes of the African trypanosome in Escherichia coli.
The African trypanosome, Trypanosoma brucei, contains multiple genes for both alpha- and beta-tubulins, which code for similar if not identical proteins. Studies of the structure and function of trypanosome microtubules have been limited due to the difficulties in obtaining sufficient amounts of purified tubulin. To produce large amounts of purified tubulin for studies of structure and function and to begin developing a system for producing systematic alterations of tubulin structure we have cloned and expressed the alpha- and beta-tubulin genes of T. brucei in Escherichia coli to produce the unfused proteins. Controlled high-level expression of both alpha- and beta-tubulin was achieved using a plasmid vector, pOTS, in which expression is controlled by phage lambda promoter/operator and a temperature-sensitive lambda repressor. The tubulins produced are insoluble, as has been found for many other proteins expressed to high levels in E. coli; they are readily purified to near homogeneity by chromatography on DEAE-cellulose in 7 M urea. N-terminal analysis of the purified proteins indicates that they are initiated correctly and that the N-formyl group is removed from the initiating methionine. This factor will probably prove important in the reconstitution of biologically active tubulin.
Wu
J
J
Department of Biochemistry, University of Kansas Medical Center, Kansas City 66103.
Yarbrough
L R
LR
D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D010957:Plasmids; D014346:Trypanosoma brucei brucei / Q000235:genetics*; D014404:Tubulin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D014404:Tubulin