title: Rapid purification of recombinant proteins fused to chicken avidin.
abstract: A novel expression vector (pAVEX16C) has been constructed that directs the synthesis of desired polypeptides as fusions with the C terminus of chicken egg-white avidin (Avd). With this and a commercial GST gene (encoding glutathione S-transferase) fusion vector (pGEX-3X, Pharmacia), we produced Avd as fusions C- and N-terminally linked to GST in Escherichia coli. By using the Avd tail and a simple affinity purification protocol, including biotin-agarose, we were able to obtain 1-2 micrograms/ml of highly purified Avd::GST and GST::Avd from crude bacterial lysates. The produced proteins were, to a great extent, in soluble fraction when the cells were grown at 22 degrees C and disrupted with a detergent, N-laurylsarcosine. The fusion proteins could also be affinity-purified with the GST tail using glutathione-Sepharose 4B, but the yield of GST::Avd was significantly lower than when using the Avd tail. Our results therefore indicate that it is possible to produce, in E. coli, biologically active fusion proteins consisting of Avd C- or N-terminally linked with the desired protein which then can easily be purified by a simple affinity chromatography procedure.
authors.affiliation: Department of Biological and Environmental Science, University of Jyväskylä, Finland.
authors.forename: K J
authors.initials: KJ
authors.lastname: Airenne
authors.forename: M S
authors.initials: MS
authors.lastname: Kulomaa
mesh_terms: D000595:Amino Acid Sequence; D001360:Avidin / Q000737:chemistry*; D001483:Base Sequence; D004926:Escherichia coli; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*
chemical_list: D011993:Recombinant Fusion Proteins; D001360:Avidin; D005982:Glutathione Transferase