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Expression and Purification of Wild Type and Variant Ribonuclease Inhibitor Proteins
Author(s) -
Prophet Sarah,
Uebersohn Alec,
Dods Galen,
Dickson Kimberly
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.882.13
Subject(s) - ribonuclease , cysteine , biochemistry , function (biology) , biology , angiogenin , s tag , microbiology and biotechnology , ribonuclease iii , chemistry , rna , enzyme , genetics , gene , rna interference , angiogenesis
The ribonuclease inhibitor (RI) is an intracellular mammalian protein that binds select members of the vertebrate‐specific ribonuclease superfamily. The interaction between RI and its target ribonucleases is one of the tightest non‐covalent interactions to be discovered and involves extensive contacts between the two proteins that block the ribonucleolytic activity of the target as well as its ability to interact with other proteins. Through these tight protein‐protein interactions, RI regulates the functions of its target ribonucleses, which influence a wide variety of cellular and physiological processes including blood vessel growth, maintenance of neuronal health, innate immunity, and responses to cell stress. RI also interacts with proteins outside the vertebrate‐specific ribonuclease superfamily such as Drosha and PTEN to regulate other molecular pathways. A defining characteristic of RI is its extreme oxidation sensitivity, a result of its unusually high cysteine content. Oxidation of any of the 32 reduced cysteines in RI facilitates rapid denaturation and inactivation. The goal of this work is to understand the role of cysteine residues in the structure and function of RI. We have constructed 7 variants of RI in which a subset or all of the cysteines have been conservatively replaced. Here, we report a novel approach for the expression and purification for this library of RI proteins that does not depend upon affinity purification with ribonucleases.