Identification and isolation of human insulin A and B chains by high-performance liquid chromatography.
A method for the isolation, identification and quantification of human insulin A and B chains by high-performance liquid chromatography (HPLC) is described. These chains were isolated from a peptide mixture produced by E. coli with modified genes obtained by genetic engineering. The method is based on the use of hydrophilic reagents, forming ion pairs in a reversed-phase column. Because some undesirable effects resulting from the use of phosphoric acid were observed, especially with the B chain, a new HPLC method was developed for each of the two human insulin chains. The use of trifluoroacetic acid as a counter ion for the A chain and of formic acid for the B chain led to the rapid isolation and purification of each chain by HPLC. The advantage of this method is that it provides a highly pure product, which was identified by polyacrylamide gel electrophoresis and amino acid analysis.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D010455:Peptides / Q000032:analysis*; D011994:Recombinant Proteins
D000596:Amino Acids; D007328:Insulin; D010455:Peptides; D011994:Recombinant Proteins