A novel method for the production of fully modified K-Ras 4B.
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.
Department of Medicinal Chemistry, College of Pharmacy, University of Illinois at Chicago, Chicago, IL, USA.
Tanmay S
TS
Chavan
James O
JO
Meyer
Luke
L
Chisholm
Malgorzata
M
Dobosz-Bartoszek
Vadim
V
Gaponenko
D005818:Genetic Engineering / Q000379:methods*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D017368:Protein Prenylation; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance
D006160:Guanosine Triphosphate; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras)