Functional
Di-domain of Neuronal Growth Inhibitory Factor
JI Qing-Zhou, KANG Qiao-Hua, ZHOU
Yan-Jiao,REN Hong-Wei, RU Bing-Gen*
( National Laboratory of Protein Engineering, College of Life
Sciences, Peking University, Beijing 100871, China )
Abstract 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 β-domain being functional. GIF functional di-domain
(GIFβ-β) was constructed to study the structure and function of GIF. N terminal
β-domain and C terminal β-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 GIFβ-β. Circular dichroism (CD) spectroscopy
show GIFβ-β has characteristic metal-sulfhydryl clusters of metallothionein
family. Inhibitory activities detected by the MTT reduction assay are: GIF >
GIFβ-β > GIF β-domain.
Key words neuronal growth inhibitory factor; β-domain;
functional di-domain; expression; function
*Corresponding author: Tel,
86-10-62751842; Fax, 86-10-62751842; e-mail, [email protected]