Immobilized insulin for high capacity affinity chromatography of insulin receptors.

J Markussen,J Halstrøm,F.C Wiberg,L Schäffer

doi:10.1016/s0021-9258(18)55136-x

Abstract

Insulin receptors can be purified by affinity chromatography on immobilized insulin, but published methods all suffer from a rather low capacity of the affinity columns. By using insulin that has been protected in positions A1 and B29, we have been able to couple the insulin selectively through the B1 amino group to divinyl sulfone-activated agarose. The N terminus of the B-chain is the most innocuous site as far as receptor-insulin interaction is concerned, and this strategy allowed us to make affinity columns with capacities of several milligrams of receptor/ml of resin. The receptor used was the soluble ectodomain of the human insulin receptor, produced in transfected baby hamster kidney cells. The column preparation and the elution conditions are described in detail, as the efficacy of the purification depends strongly on both. The purity of the eluted receptors was so high that quantitative amino acid analysis fitted with theory. The molar absorption coefficient at 278.5 nm was 296,000 M-1 cm-1. Finally, it could be unequivocally established that the soluble receptor binds two molecules of insulin with equal affinity.

Highlights

Insulin receptors can be purified by affinity chromatography on immobilized insulin, but published methods all suffer from a rather low capacity of the affinity columns
Fujita-Yamaguchi et al (1983) used the same spacer that the soluble receptor binds two molecules of insulin anchored to Sepharose 4B and coupled the N-hydroxysucciwith equal affinity
In the experiments where the glycine spacer was omitted, x = 0, the Boc2 porcine insulin dissolved in Me2S0 was added directly to the reduce the potency in the free fat cell assay to about 20-30 Mini-Leak suspensionas described above

Summary

Introduction

Insulin receptors can be purified by affinity chromatography on immobilized insulin, but published methods all suffer from a rather low capacity of the affinity columns. In the experiments where the glycine spacer was omitted, x = 0, the Boc2 porcine insulin dissolved in Me2S0 was added directly to the reduce the potency in the free fat cell assay to about 20-30 Mini-Leak suspensionas described above.

Results

Conclusion