[Functional di-domain of neuronal growth inhibitory factor]

Neuronal growth inhibitory factor (GIF), known also as metallothionein-III (MT-III), was the first validated to be capable of inhibiting growth of neuronal cells in nervous system, its beta-domain being functional. GIF functional di-domain (GIFbeta- beta) was constructed to study the structure and function of GIF. N terminal beta-domain and C terminal beta-domain cDNAs were amplified by PCR, inserted into vector pGEX-4T-1 and expressed in Escherichia coli, as carboxyl terminal extension of glutathione-S-transferase (GST), by IPTG induction. After digestion by thrombin, the fusion protein was isolated by passing through a glutathione-Sepharose 4B affinity chromatography column and was purified by gel fit ration on Sephacryl-S100. About 60 mg protein per liter of bacterial cell culture was achieved. The results of SDS-PAGE, amino acid composition, molecular mass, the ratio of metal/protein and sulfhydryl group/protein showed that the purified protein was the GIFbeta- beta. Circular dichroism (CD) spectroscopy show GIFbeta- beta has characteristic metal-sulfhydryl clusters of metallothionein family. Inhibitory activities detected by the MTT reduction assay are: GIF > GIFbeta-beta > GIF beta-domain.