Protein prenylation in an insect cell-free protein synthesis system and identification of products by mass spectrometry

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 a...

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Bibliographic Details
Published in:Proteomics (Weinheim) 2007-06, Vol.7 (12), p.1942-1950
Main Authors: Suzuki, Takashi, Ito, Masaaki, Ezure, Toru, Shikata, Masamitsu, Ando, Eiji, Utsumi, Toshihiko, Tsunasawa, Susumu, Nishimura, Osamu
Format: Article
Language:English
Subjects:
Alkyl and Aryl Transferases - metabolism
Analytical, structural and metabolic biochemistry
Animals
Biological and medical sciences
Cell Extracts
Cloning, Molecular
Farnesyltranstransferase - metabolism
Fundamental and applied biological sciences. Psychology
Gelsolin - metabolism
Humans
Insect cell-free protein synthesis system
Insect Proteins - metabolism
Insecta - metabolism
MALDI-quadrupole-IT-TOF MS
MALDI-TOF MS
Miscellaneous
Protein Prenylation
Proteins
Rabbits
Rats
rho GTP-Binding Proteins - metabolism
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Summary: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.
ISSN:1615-9853
1615-9861
DOI:10.1002/pmic.200700237