Identification of Anchor Points for Chemical Modification of a Small Cysteine-Rich Protein by Using a Cysteine Scan
| Autor: | Tine N. Vinther, Thomas Kjeldsen, Thomas Åskov Pedersen, Ulla Ribel, Knud J. Jensen, Frantisek Hubalek |
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| Rok vydání: | 2011 |
| Předmět: |
Male
Alkylation Saccharomyces cerevisiae Biochemistry Polyethylene Glycols Maleimides chemistry.chemical_compound Residue (chemistry) Tandem Mass Spectrometry Animals Insulin Moiety Cysteine Disulfides Rats Wistar Molecular Biology Maleimide Chromatography High Pressure Liquid biology Chemistry Organic Chemistry Chemical modification Stereoisomerism Biological activity biology.organism_classification Rats Molecular Medicine |
| Zdroj: | ChemBioChem. 12:2448-2455 |
| ISSN: | 1439-4227 |
| DOI: | 10.1002/cbic.201100464 |
| Popis: | Chemical modifications of proteins are increasingly important in the development of protein drugs with fine-tuned properties. Regioselective modification, such as the chemoselective alkylation of an unpaired cysteine residue, is a prerequisite for obtaining homogenous protein products. The introduction of an unpaired Cys into the Cys-rich protein, insulin, was investigated by using a Cys scan. This was challenging as the introduced Cys could interfere with insulin's three existing disulfide bonds. However, eight insulin precursors were expressed in Saccharomyces cerevisiae with good yields. Although extensive post-translational modifications of the unpaired Cys were observed, the majority could be removed by selective reduction. An example Cys(7) insulin analogue was modified with a PEGylated maleimide moiety. The new variant was active in in vitro and in vivo models. Our results show that even small Cys-rich proteins can be expressed with additional unpaired Cys in meaningful yields and further chemically modified, while maintaining their biological activity. |
| Databáze: | OpenAIRE |
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