Hydroxyl group of insulin A19Tyr is essential for receptor binding: studies on (A19Phe)insulin.
One mutant proinsulin gene was constructed through PCR with the code of A19Tyr changed into that of Phe. The mutant proinsulin, (A19Phe)-lysproinsulin (F19KPI) was expressed in E. coli and purified. After trypsin and carboxypeptidase B cleavage and Resource Q separation, (A19Phe)-human insulin (F19HI) was obtained. With native insulin as standard, activity assay and structural analysis were carried out. It was found that the conformation of F19HI is very similar to that of native insulin according to the data from native PAGE, reverse-phase FPLC and circular dichroism spectrum, which are also in agreement with the result of radioimmune assay. F19HI retains almost full immune activity, but displays only 13.3% of receptor binding activity. These results suggest that the hydroxyl group of insulin A19Tyr is essential for receptor binding.
National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing, P.R. China.
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Tang
D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006860:Hydrogen Bonding; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011863:Radioimmunoassay; D011972:Receptor, Insulin / Q000378:metabolism*
D007328:Insulin; C027602:insulin, Phe(A19)-; D011384:Proinsulin; D011972:Receptor, Insulin