Characterization of factors favoring the expression of soluble protozoan tubulin proteins in Escherichia coli.
The alpha- and beta-tubulin genes of the parasitic protozoa Giardia duodenalis, Cryptosporidium parvum, and Encephalitozoon intestinalis have been overexpressed in soluble form using Escherichia coli-based expression systems. Several expression systems were compared in terms of the amount of soluble protein produced with different fusion partners, strains of E. coli BL21, and expression temperatures. The cleavability of the fusion partner was also assessed in terms of post-expression applications of the recombinant protein. The maltose-binding protein (MBP) and glutathione S-transferase (GST) fusion partners produced the highest expression levels for all six proteins without the formation of inclusion bodies. The expression system also provided a means of purifying the soluble protein using affinity and anion-exchange chromatography while minimizing protein losses. The yield and purity were therefore very high for both the MBP and GST systems. The tubulin monomers were demonstrated to be assembly-competent using a standard dimerization assay and also retained full antigenicity with monoclonal antibodies. This study presents several methods which are suitable for producing soluble tubulin monomers and, thus, circumventing the formation of inclusion bodies which necessitates re-folding of the tubulin.
Division of Veterinary and Biomedical Sciences, Murdoch University, South Street, Perth, WA 6150, Australia. lmacdona@central.murdoch.edu.ac
Louisa M
LM
MacDonald
Anthony
A
Armson
R C Andrew
RC
Thompson
James A
JA
Reynoldson
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003062:Codon; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism*; D005872:Giardia; D005982:Glutathione Transferase / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature; D014404:Tubulin / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003062:Codon; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D005982:Glutathione Transferase