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Expression and Purification of Cdc42 and PBD46
Author(s) -
Evans Edward J,
Adams Paul D
Publication year - 2012
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.26.1_supplement.973.4
Subject(s) - cdc42 , guanine nucleotide exchange factor , effector , gtpase , microbiology and biotechnology , threonine , ras superfamily , gtpase activating protein , biochemistry , serine , chemistry , gtp' , phosphorylation , mutant , signal transduction , biology , g protein , enzyme , gene
The Ras superfamily consists of low molecular weight G‐proteins that are heavily involved in cellular signaling, leading to various downstream responses. Like other Ras proteins, Cdc42 acts as a molecular switch interchanging from its active (GTP‐bound) and inactive (GDP‐bound) state through the utilization of GAP (GTPase‐activating proteins) and GEF (guanine nucleotide exchange factors) effectors. It has been shown that Cdc42 signaling pathways play a major role in cell proliferation and differentiation, leading to tumor growth. PBD46 is an effector of Cdc42 proven to inhibit GTP hydrolysis, stabilizing the Ras protein in its active state. PBD46, as well as many other effectors, bind to the Switch I region (amino acids 28–40) of Cdc42. Threonine of position 35 was replaced with alanine forming a Cdc42 mutant. With the use of affinity column chromatography, Cdc42(WT), Cdc42(T35A), and PBD46 were expressed and purified for future tests of the Switch I binding interface.