Immobilized insulin for high capacity affinity chromatography of insulin receptors.
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.
Markussen
J
J
Novo Research Institute, Bagsvaerd, Denmark.
Halstrøm
J
J
Wiberg
F C
FC
Schäffer
L
L
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D007328:Insulin / Q000378:metabolism*; D011972:Receptor, Insulin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D007328:Insulin; D011972:Receptor, Insulin