Molecular identification and characterization of camel milk insulin

The number of people suffering from Diabetes mellitus in both developed and developing nations has increased dramatically over the past two-three decades and this increase is predicted. The condition can result in numerous complications in the patients suffering from the disease if it is not with long-term effects including retinopathy with potential blindness, nephropathy that may lead to renal failure, and/or neuropathy and an increased risk of cardiovascular, peripheral vascular and cerebrovascular disease. Camel milk consumption has been reported to have a positive effect with respect to diabetic severity and complications and as a treatment of diabetes with regular camel milk drinkers requiring lower doses of insulin to control their blood glucose levels. A number of studies have now suggested a direct link between camel milk insulin and the effect of drinking camel milk on diabetic suffers. In this study, insulin from dromedary camel (Camelus dromedaries) milk was isolated and characterized in order to investigate the stability and nature of the protein to begin to understand why it may have positive benefits on patients consuming camel milk Initia1ly the camel insulin gene was amplified and sequenced from 32 camels using primers used to amplify the camel pro insulin cDNA. The sequenced fragment was aligned using a BLAST search and the sequence matched a sequence from lama with 96% homology. A number of SNPs were identified in key regions of the camel gene sequence, although none in the coding sequence with 3 located in the C-peptide intron, 2 in the 3' -UTR and 1 after the termination signal. When the gene sequence was converted to the corresponding amino acid sequence this

revealed that across the 13 species compared the camel insulin has two unique amino_ acid changes in the signal peptide, one unique amino acid change in the B-chain and two changes in the A-chain compared to human (which are observed in some other species). There is much variation across species in the C-peptide region and in the camel there were various changes compared to other species. Using the sequence, 3D homology modeling of camel proinsulin was undertaken which suggests the formation of different secondary structure compared to that for human insulin which may impact upon the stability of the molecule. Using FISH, the camel insulin gene was also mapped to the distal (Telomeric) end of the q-arm of camel chromosome 10. In addition to investigating camel insulin at the genomic level, studies were undertaken at the protein level. For protein analysis, fresh camel milk samples were collected from the Emirates dairy farm. The samples were initially defatted and then treated with ethanol in order to precipitate casein which is the major milk protein and other large molecular weight proteins, before the supernatant was passed through an ion-exchange chromatography column of A-25 DEAE Sephadex beads. The bound camel milk insulin was then eluted and further purified by immune-affinity chromatography using an anti -human insulin antibody. The resulting insulin concentration and activity was measured by radioimmunoassay using a human insulin kit. The camel insulin gene was also cloned into a mammalian expression vector and transfected into a mammalian cell line and attempts made to produce cell lines stably expressing camel insulin. The thermostability of the insulin purified from camel milk was then compared with human and bovine insulin in camel and bovine milk. The results suggest that camel insulin appears to be stabilised due to the unique both amino acid variants in comparison to human insulin but these studies also suggested that camel milk itself provides protection against thermostability which bovine milk does not and this property contributed the most towards the enhanced thermostability of camel milk insulin. The exact nature of this protection and the agents responsible are not currently known, but further elucidation of the mechanism(s) may provide possible routes for the thermostabilisation of other proteins.