|
In order to study the structure-function relationships of α-
bungarotoxin (α-BuTx) we have constructed the cDNAs encoding
α-BuTx and its isoform from the cellular RNA isolated from the
venom glands of Bungarus multicinctus by RT-PCR. The cDNAs were
cloned into the pGEX-2T vectors and expressed in E.coli as the
fusion proteins. The fusion proteins were isolated by
glutathion-S-transferase(GST)-affinity column. After thromin
cleavage , the recombinant α-BuTx and its isoform were purified
by RP-HPLC. The recombinant α-BuTx and its isoform contain
additional 7 and 5 amino acid residues linked to the N- and C-
terminal ends of toxin molecule , respectively. The isoform
exhibits a high sequence identity (98%) to α-BuTx with
substitution of Thr-6 for Pro and Arg-36 for Lys in amino acid
sequence. Both recombinant proteins showed the same antigenicity
as native α-BuTx by radioimmunoassay. Although both recombinant
toxins were capable of binding to the nicotinic acetylcholine
receptor (nAChR) , but the apparent affinity which was reduced
to about 10% that of native α-BuTx. The results reveal that the
substitutions of Thr-6 for Pro and Arg-36 for Lys do not affect
the antigenicity and nAChR-binding activity. The improvement of
nAChR-binding activity of the recombinant toxins by protein
refolding and removal of the additional amino acid residues
linked to the α-BuTx molecule will be discussed.
|