Inhibition of platelet aggregation of a mutant proinsulin molecule engineered by introduction of a native Arg-Gly-Asp sequence.
A 13 amino acid sequence, CRVARGDWNDNYC, originated from disintegrin eristostatin, was introduced into an inactive human proinsulin molecule between the B29 and A2 sites to replace proinsulin C-peptide by molecular cloning techniques. The constructed Arg-Gly-Asp (RGD)-proinsulin gene was cloned into a temperature-inducible vector pBV220 and expressed in Escherichia coli. The expressed RGD-proinsulin was refolded and purified by Sephadex G50 and DEAE-Sephadex A25 separations. The chemical identity was confirmed by both amino acid composition and mass spectrometry analyses. This RGD-proinsulin showed an inhibitory activity of adenosine 5'-diphosphate-induced human platelet aggregation with an IC50 value of 200 nM. Its insulin receptor binding activity remained as low as 0.03% with native insulin as a control, and its insulin immune activity retained 27.6% compared with proinsulin.
Yang
Z H
ZH
National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China.
Jing
J
J
Tang
J G
JG
D000244:Adenosine Diphosphate / Q000494:pharmacology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D002096:C-Peptide / Q000235:genetics; D002942:Circular Dichroism; D019483:Disintegrins / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli; D006801:Humans; D020128:Inhibitory Concentration 50; D007328:Insulin / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D016254:Mutagenesis, Insertional / Q000235:genetics; D009842:Oligopeptides / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D010974:Platelet Aggregation / Q000187:drug effects*; D011384:Proinsulin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D015202:Protein Engineering; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D011972:Receptor, Insulin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D014757:Viper Venoms / Q000737:chemistry / Q000235:genetics
D002096:C-Peptide; D019483:Disintegrins; D007328:Insulin; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014757:Viper Venoms; C084060:eristostatin; D000244:Adenosine Diphosphate; C047981:arginyl-glycyl-aspartic acid; D011384:Proinsulin; D011972:Receptor, Insulin