A novel expression vector (pAVEX16C) has been constructed that directs
the synthesis of desired polypetides as fusions with the C terminus o
f 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 Esc
herichia coli. By using the Avd tail and a simple affinity purificatio
n protocol, including biotin-agarose, we were able to obtain 1-2 mu g/
ml of highly purified AVd::GST and GST::AVd from crude bacterial lysat
es. The produced proteins were, to a great extent, in soluble fraction
when the cells were grown at 22 degrees C and disrupted with a deterg
ent, N-laurylsarcosine. The fusion proteins could also be affinity-pur
ified 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-termina
lly linked with the desired protein which then can easily be purified
by a simple affinity chromatography procedure.