Clone And Preliminary Functional Analysis Of Human CRBN Gene

From our large cDNA cloning and sequencing plan, by cDNA microarray analysis together with bioinformatics analysis, we selected CRBN (cereblon) gene which is related with diseases as follows for further research.Mental retardation (MR) has a strong impact on population quality. The research of MR is rare while the research of mild mental retardation which has the highest incidence of MR is rarer. A genetic or inherited metabolic etiology is implicated in two-thirds of mental retardation cases. CRBN is the first found to be involved in autosomal recessive nonsyndromic mental retardation (ARNSMR) and has relativity with some dominant MR. After being separated, the human CRBN gene which spanned more than 29 Kb of genomic DNA located to human chromosome between 3p26.2 by bioinformatics analysis. We also found CRBN had the N- terminal of an ATP-dependent LON domain, which belongs to ATP-dependent LON protease family.The result of RT-PCR showed that CRBN were expressed at an extremely high level in human testis. It also highly expressed in spleen, prostate, liver, pancreas, placenta and kidney and moderately expressed in lung, skeletal muscle, ovary, small intestine, peripheral blood leukocyte, colon and brain and there were no expression in heart and thymus. Its high expression in testis suggested the important role of CRBN in organogenesis. RCRBN was reported to have the direct interaction with the cytosolic carboxy-terminus of the large conductance Ca²⁺-activated K⁺ (BK_Ca) channel a subunit as a binding protein. As expression of large conductance calcium-activated potassium channels decreases cellular protein tyrosine phosphorylation, CRBN might effect an important signal transduction pathways. Since CRBN was similar to rCRBN (amino acid identities of 93%), it was suggested that CRBN might also be involved in signal pathways, not only related to memory and long-termpotentiation.Constructing normal CRBN and mutation CRBN with the pEGFP-C1 plasmid separately, GFP location of CRBNgene product shows both proteins were localized in the whole AD293 cells. CRBN was not only related with mitochondria where CRBN participated in energy metabolism but also related with cytoplasm and nucleus. We speculate that CRBN may participate in other biological processes.Normal CRBN and mutation CRBN fusion protein which were constructed with the entire prokaryotic GST protein respectively were induced by IPTG. After normal CRBN fusion protein was purified affinitively, the results of SDS-PAGE showed proteins purified were not single, which suggested the non-target proteins might be interacted with CRBN protein or degraded by CRBN. These unknown protein have not researched yet.