The ability to perform site-specific protein modifications is a valuable tool used for study and isolation of proteins of interest.  In particular, introducing functional groups can allow for reversible protein immobilization, and provides an efficient and scalable method for protein isolation.  For example, several labs have exploited protein farnesyltransferase (FTase) to site-specifically label proteins via prenylation with chemically modified forms of natural farnesyl isoprenoid pyrophosphate (FPP).  However, due to the potential for cross-reactivity with endogenous proteins, FTase-catalyzed labeling of specific target proteins has been limited to proteins expressed in organisms which lack endogenous protein prenylation, such as bacteria. To expand upon this approach, we have explored the use of engineered FTase variants selected for their ability to prenylate substrate sequences that are unreactive with wild type FTase. Combining these engineered FTase variants with functionalized FPP analogs yields a two-stage bioorthogonal selective technique for isolating target proteins, even in the presence of natural prenyltransferase substrate proteins in eukaryotic cell lysates.  This method is demonstrated by selective isolation of model fluorescent proteins in bacterial lysates in the presence of competitor proteins, followed by extension into target protein isolation from eukaryotic lysates.  The selectivity of prenylation, the minimal size of the four amino acid C-terminal FTase recognition motif, and the potential for a range of bioorthogonal chemical labeling reactions combine to provide a unique advantage to this approach. The ability to rapidly label and isolate proteins from crude lysates in the presence of other natural prenyltransferase substrates makes this approach applicable across multiple systems, and may facilitate investigation of a wide range of target proteins which may be otherwise difficult to isolate.

Chemoenzymatic protein labeling and isolation from eukaryotic cell lysates using prenyltransferases with reengineered substrate selectivity

251st National Meeting (2016)

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