Effects of Cysteine to Serine Substitutions in the Two Inter-Chain Disulfide Bonds of Insulin
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Zhan-Yun Guo
Abstract
Using sitedirected mutagenesis we deleted the two interchain disulfide bonds of insulin, separately or both, by substitution of the cysteine residues with serine. Deletion of A20-B19 or both of the two interchain disulfide bonds resulted in the complete loss of secretion of the mutant singlechain porcine insulin precursor (PIP) from Saccharomyces cerevisiae cells. Removal of the A7-B7 disulfide bond resulted in a large reduction of secretion, but we could obtain the mutant for analysis of its biological and some physico chemical properties. The A7-B7 disulfide bond deleted insulin mutant retained only 0.1% receptorbinding activity compared with porcine insulin, and its in vivo biological potency measured by mouse convulsion assay was also very low. We also studied some physicochemical properties of the mutant using circular dichroism, native polyacrylamide gel electrophoresis and reversedphase HPLC, which revealed some structural changes of the mutant peptides compared to native insulin. The present study shows that the two interchain disulfide bonds are important for efficient in vivo folding/secretion of PIP from yeast, especially the A20-B19 disulfide bond, and that the A7-B7 disulfide bond is crucial for maintaining the native conformation and biological activity of insulin.
Copyright © 2001 by Walter de Gruyter GmbH & Co. KG
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- Fluorescence Correlation Spectroscopy as a Tool to Investigate Single Molecule Probe Dynamics in Thin Polymer Films
- Ligand-Receptor Interactions in the Membrane of Cultured Cells Monitored by Fluorescence Correlation Spectroscopy
- Fluorescence Fluctuation Analysis for the Study of Interactions between Oligonucleotides and Polycationic Polymers
- Determination of the Net Exchange Rate of Tubulin Dimer in Steady-State Microtubules by Fluorescence Correlation Spectroscopy
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