Merck
  • Home
  • Search Results
  • Recombinant canine single chain insulin analogues: insulin receptor binding capacity and ability to stimulate glucose uptake.

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

Veterinary journal (London, England : 1997) (2014-12-03)
Jamie P Adams, Angela L Holder, Brian Catchpole
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium dodecyl sulfate, 92.5-100.5% based on total alkyl sulfate content basis
Sigma-Aldrich
Hydrochloric acid, 36.5-38.0%, BioReagent, for molecular biology
Sigma-Aldrich
Sodium dodecyl sulfate, ReagentPlus®, ≥98.5% (GC)
Sigma-Aldrich
3,3′,5,5′-Tetramethylbenzidine, ≥98% (TLC)
Sigma-Aldrich
3,3′,5,5′-Tetramethylbenzidine, tablet, 1 mg substrate per tablet
Sigma-Aldrich
3,3′,5,5′-Tetramethylbenzidine, ≥99%
Sigma-Aldrich
Sodium dodecyl sulfate, BioReagent, suitable for electrophoresis, for molecular biology, ≥98.5% (GC)
Sigma-Aldrich
Sodium dodecyl sulfate, BioXtra, ≥99.0% (GC)
Sigma-Aldrich
Hydrochloric acid solution, 1.0 N, BioReagent, suitable for cell culture
Supelco
Sodium dodecyl sulfate, suitable for ion pair chromatography, LiChropur, ≥99.0%
Sigma-Aldrich
Sodium dodecyl sulfate, BioUltra, for molecular biology, ≥99.0% (GC)
Sigma-Aldrich
Sodium dodecyl sulfate, ACS reagent, ≥99.0%
Supelco
Hydrogen chloride – ethanol solution, ~1.25 M HCl, for GC derivatization, LiChropur
Sigma-Aldrich
Sulfuric acid, 99.999%
Sigma-Aldrich
6-Benzylaminopurine, ReagentPlus®, ≥99.0% (HPLC)
Sigma-Aldrich
Sodium dodecyl sulfate, ≥98.0% (GC)
Sigma-Aldrich
3,3′,5,5′-Tetramethylbenzidine, ≥98.0% (NT)
Supelco
Sulfuric acid, for the determination of nitrogen, ≥97.0%
Sigma-Aldrich
Hydrochloric acid solution, ~6 M in H2O, for amino acid analysis
Supelco
Hydrogen chloride – 2-propanol solution, ~1.25 M HCl (T), for GC derivatization, LiChropur
Supelco
3,3′,5,5′-Tetramethylbenzidine, standard for GC
Sigma-Aldrich
6-Benzylaminopurine, suitable for plant cell culture
Supelco
Hydrochloric acid solution, volumetric, 0.1 M HCl (0.1N), endotoxin free
Sigma-Aldrich
Ampicillin, meets USP testing specifications
Sigma-Aldrich
Sodium dodecyl sulfate, ≥90%
Sigma-Aldrich
Sodium dodecyl sulfate, tested according to NF, mixture of sodium alkyl sulfates consisting mainly of sodium dodecyl sulfate
Sigma-Aldrich
Sodium dodecyl sulfate, BioReagent, suitable for electrophoresis, for molecular biology, ≥98.5% (GC), free-flowing, Redi-Dri
Sigma-Aldrich
Hydrogen chloride solution, 4.0 M in dioxane
Sigma-Aldrich
Hydrochloric acid, 37 wt. % in H2O, 99.999% trace metals basis
Sigma-Aldrich
Hydrochloric acid, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., fuming, ≥37%, APHA: ≤10