Abstract
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.
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Yang, ZH., Jing, J. & Tang, JG. Inhibition of platelet aggregation of a mutant proinsulin molecule engineered by introduction of a native Arg-Gly-Asp sequence. Appl Biochem Biotechnol 90, 1–10 (2001). https://doi.org/10.1385/ABAB:90:1:1
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DOI: https://doi.org/10.1385/ABAB:90:1:1