Effects of deleting A19 tyrosine from insulin.
A mutant proinsulin gene was constructed through PCR mediated mutagenesis. The code of A19Tyr was deleted. The mutant proinsulin, (deltaY)19-lys-proinsulin [(deltaY)19KPI], was expressed in E. coli and purified. After treatment with trypsin and carboxypeptidase B, and Resource Q separation, (deltaY)19-human insulin [(deltaY)19HI] was obtained. It retains 63.6% of receptor binding activity but only 2.2% of immune activity, and shows a longer retaining time on reverse-phase FPLC and a slower mobility by native PAGE analysis. These results suggest that the deletion of A19Tyr causes some conformational changes on insulin, which plays a minor role on the affinity of insulin to its receptor, and a major role on immunogenicity of the hormone.
Du
X
X
National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing, PR China.
Tang
J G
JG
D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007328:Insulin / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D011384:Proinsulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D014357:Trypsin; D014443:Tyrosine
D007328:Insulin; D011994:Recombinant Proteins; D014443:Tyrosine; D011384:Proinsulin; D014357:Trypsin