Domain splicing within metallothionein family

Metallothioneins(MTs) fold into two separate domains: beta - domain and alp ha - domain which obviously differ in function. Neuronal growth inhibitory factor (GIF), named as Metallothionein - III ( MT - III), is first characte rized to be capable of inhibiting the growth of neuronal cell in nervous sy stem, and beta - domain is its functional domain. To study deep the structu re and function of MTs, GIF and their domains; we construct the splicing - domains within metallothioneins family, beta (GIF) - alpha (MT- 1) (beta (I II) - alpha (I)) and beta (MT - I) - alpha (GIF) (beta (I) - alpha (III)) c DNAs were amplified by polymerase chain reaction (PCR), inserted into vecto r pGEX - 4T - 1, expressed in Escherichia coli as carboxyl terminal extensi on of glutathione - S - transferase ( GST) by IPTG's induction. After the f usion protein had been digested by thrombin on a Glutathione - Sepharose 4B affinity chromatography column, recombinant beta (III) - alpha (I) and bet a (I) - alpha (III) were purified by gel fitration on Sephacryl - S100. Eig hty mg of the protein can be obtained from every liter medium after ferment ation. The results of SDS - PAGE, amino acids composition, molecular mass, the ratio of metal/protein and sulfhydryl group/protein confirm that the pu rified protein is the desired one. Ultraviolet ( UV) absorption spectroscop y and circular dichroism ( CD) spectroscopy show splicing - domains have th e characteristic metal - sulfhydryl group clusters of metahothionein family . beta (III) - alpha (I), similar with GIF, displays the neuronal growth in hibitory activity.