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Protein prenylation in an insect cell‐free protein synthesis system and identification of products by mass spectrometry
Author(s) -
Suzuki Takashi,
Ito Masaaki,
Ezure Toru,
Shikata Masamitsu,
Ando Eiji,
Utsumi Toshihiko,
Tsunasawa Susumu,
Nishimura Osamu
Publication year - 2007
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200700237
Subject(s) - prenylation , farnesyltransferase , prenyltransferase , geranylgeranylation , biochemistry , geranylgeranyl pyrophosphate , farnesyl pyrophosphate , reticulocyte , cell free protein synthesis , chemistry , tandem mass spectrometry , cell free system , biology , protein biosynthesis , mass spectrometry , biosynthesis , enzyme , rna , chromatography , gene
To evaluate the ability of an insect cell‐free protein synthesis system to carry out proper protein prenylation, several CAIX (X indicates any C‐terminal amino acid) sequences were introduced into the C‐terminus of truncated human gelsolin (tGelsolin). Tryptic digests of these mutant proteins were analyzed by MALDI‐TOF MS and MALDI‐quadrupole‐IT‐TOF MS. The results indicated that the insect cell‐free protein synthesis system possesses both farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase) I, as is the case of the rabbit reticulocyte lysate system. The C‐terminal amino acid sequence requirements for protein prenylation in this system showed high similarity to those observed in rat prenyltransferases. In the case of rhoC, which is a natural geranylgeranylated protein, it was found that it could serve as a substrate for both prenyltransferases in the presence of either farnesyl or geranylgeranyl pyrophosphate, whereas geranylgeranylation was only observed when both prenyl pyrophosphates were added to the in vitro translation reaction mixture. Thus, a combination of the cell‐free protein synthesis system with MS is an effective strategy to analyze protein prenylation.