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Expression, Purification, and Crystallization of the K‐Ras Q61L and D92Y mutations
Author(s) -
Henao Jhonatan,
Parker Jillian,
Mattos Carla
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.893.10
Subject(s) - gene isoform , gtpase , gtp' , mutant , mutagenesis , aspartic acid , biochemistry , tyrosine , lac operon , biology , escherichia coli , mutation , site directed mutagenesis , microbiology and biotechnology , chemistry , amino acid , gene , enzyme
The GTPase Ras is heavily involved in cellular signaling pathways and is implicated in 20‐30% of human cancers. It has three ubiquitous isoforms, with H‐Ras being the most heavily studied. Though the structures of H‐, K‐, and N‐Ras are very similar, studies show that mutations in each isoform occur at different frequencies, making it imperative to study each isoform individually. In particular, this research aims to obtain the crystal structures of the Q61L and D92Y mutants of K‐Ras. Site directed mutagenesis followed by a two‐staged PCR protocol was performed to successfully mutate glutamine 61 to leucine or aspartic acid 92 to tyrosine. The mutant proteins were over‐expressed using IPTG induction in an Escherichia coli system and purified via anion exchange and size exclusion chromatography methods.. A nucleotide exchange was performed to replace the GDP on K‐Ras with the GTP analogue GppNHp, so that the protein structure can be solved in its active conformation. Protein expression assays, purification, and preliminary crystallization results will be presented. This work will emphasize the inherent similarities, as well as key differences between K‐Ras mutations and its H‐Ras counterparts.