[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.
Ji
Qing-Zhou
QZ
National Laboratory of Protein Engineering, College of Life Sciences, Peking University, Beijing 100871, China. rulab@pku.edu.cn
Kang
Qiao-uaH
QU
Zhou
Yan-Jiao
YJ
Ren
Hong-Wei
HW
Ru
Bing-Gen
BG
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D000090023:Metallothionein 3; D051379:Mice; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D018076:DNA, Complementary; D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins