A Novel Method for the Production of Fully Modified K-Ras 4B
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Post-translational modifications in proteins play a major functional role. Post-translational modifications affect the way
proteins interact with each other, bind nucleotides, and localize in cellular compartments. Given the importance of post-translational
modifications in protein biology, development of methods to produce post-translationally modified proteins for biochemical
and biophysical studies is timely and significant. At the same time, obtaining post-translationally modified proteins in bacterial
expression systems is often problematic. Here, we describe a novel recombinant approach to prepare human K-Ras 4B, a protein
that is post-translationally farnesylated, proteolytically cleaved, and methylated in its C-terminus. K-Ras 4B is a member
of the Ras subfamily of small GTPases and is of interest because it is frequently mutated in human cancer.
The method relies on separate production of two structural domains—the N-terminal catalytic domain and the C-terminal peptide
chemically modified with S-farnesyl-l
-cysteine methyl ester. After the two domains are prepared, they are ligated together using the transpeptidase enzyme, sortase.
Our procedure starts with the use of the plasmid of K-Ras 4B catalytic domain containing the sortase recognition sequence.
After this, we describe the bacterial expression and purification steps used to purify K-Ras 4B and the preparation of the
conjugated C-terminal peptide. The procedure ends with the sortase-mediated ligation technique. The produced post-translationally
modified K-Ras 4B is active in a number of assays, including a GTP hydrolysis assay, Raf-1 binding assay, and surface plasmon
resonance-based phospholipid binding assay.
