Recombinant canine single chain insulin analogues: insulin receptor binding capacity and ability to stimulate glucose...

Virtually all diabetic dogs require exogenous insulin therapy to control their hyperglycaemia. In the UK, the only licensed insulin product currently available is a purified porcine insulin preparation. Recombinant insulin is somewhat problematic in terms of its manufacture, since the gene product (preproinsulin) undergoes substantial post-translational modification in pancreatic β cells before it becomes biologically active. The aim of the present study was to develop recombinant canine single chain insulin (SCI) analogues that could be produced in a prokaryotic expression system and which would require minimal processing. Three recombinant SCI constructs were developed in a prokaryotic expression vector, by replacing the insulin C-peptide sequence with one encoding a synthetic peptide (GGGPGKR), or with one of two insulin-like growth factor (IGF)-2 C-peptide coding sequences (human: SRVSRRSR; canine: SRVTRRSSR). Recombinant proteins were expressed in the periplasmic fraction of Escherichia coli and assessed for their ability to bind to the insulin and IGF-1 receptors, and to stimulate glucose uptake in 3T3-L1 adipocytes. All three recombinant SCI analogues demonstrated preferential binding to the insulin receptor compared to the IGF-1 receptor, with increased binding compared to recombinant canine proinsulin. The recombinant SCI analogues stimulated glucose uptake in 3T3-L1 adipocytes compared to negligible uptake using recombinant canine proinsulin, with the canine insulin/cIGF-2 chimaeric SCI analogue demonstrating the greatest effect. Thus, biologically-active recombinant canine SCI analogues can be produced relatively easily in bacteria, which could potentially be used for treatment of diabetic dogs.