Identification Of A Novel ERM Family Molecule And Its Functional Role In Cell Morphology

AIM: To generate polyclonal antibody of N-terminal 160 amino acids of hErmin (hErmin160), and to identify expression of hErmin in human brain and make further research about its role in cell morphology. METHODS: The coding sequence of hErmin160 was amplified by PCR from pBluescriptR plasmid encoding with full length of hErmin cDNA sequence, and then cloned into prokaryotic expression vector pET41-b(+). The plasmid was transformed into E.coli BL21(DE3)pLysS to express hErmin160 fusion protein in response to IPTG induction. The products were analyzed by SDS-PAGE and Western-blot. Expressed protein was purified by Ni2+ metal-chelating chromatograph and then injected into rabbits to generate polyclonal antibody. Blood serum after injection was purified using affinity chromatography. The polyclonal antibody was identified by Western-blot. hErmin was identified by Western-bolt and immunohistochemistry experiments, in which anti-hErmin160 antibody was used as primary antibody. After transfection, cells overexpressing hErmin were observed by immunocytochemistry experiments. RESULTS: DNA sequencing confirmed that the coding sequence of hErmin160 was completely concordant with the original sequence (BC026345, hErmin's GenBank accession number). SDS-PAGE showed that the relative molecular masses (Mr) of the expressed, purified products were about 51ku, which was in accordance with the predicted. Grayscale scanning showed that the expressed hErmin160 fusion protein accounted for 10% of the total bacteria protein in the supernatant, and the purity of purified product was 92%. Fusion protein was identified by Western-blot analysis. Blood serum after injection and purified polyclonal antibody can both bind hErmin160 specifically in Western-blot. The purified polyclonal antibody can specifically bind hErmin expressed by eukaryotic system and the expression of hErmin in human brain was confirmed. hErmin overexpression promoted cell arborization. CONCLUSION: We acquired high purification soluble hErmin160 fusion protein through the E.coli prokaryotic expression system and purified polyclonal antibody of hErmin160 protein. We have identified hErmin as a novel molecule of ERM family, which is expressed in adult human brain. hErmin overexpression promoted arborization of cultured cells.