Intein-mediated synthesis of geranylgeranylated Rab7 protein in vitro.
Production of recombinant proteins is an important prerequisite for biotechnology and life sciences in general. However, there is a paucity of methods for production of posttranslationally modified recombinant proteins or proteins with non-native functional groups, such as fluorophores, spin labels, and so forth. In this work we have used a combination of organic synthesis and in vitro protein ligation to construct monoprenylated Rab7 GTPase. The protein was prepared from a recombinant N-terminal portion and a peptide mimicking the C terminus of Rab7. For construction of a synthetic six-amino-acid-long fluorescent monoprenylated peptide, we used a block condensation strategy. Ligation was achieved with a yield of >70%. The resulting protein was purified from the unligated peptide by a combination of organic extraction and phase partitioning and refolding. The refolded monoprenylated semisynthetic Rab7 protein (Rab7GG) formed a stable complex with its natural chaperone REP-1 (Rab escort protein 1) and could serve as an acceptor of the second prenyl group in the enzymatic prenylation reaction. Using fluorescence spectroscopy, we characterized the interaction of the Rab7GG:REP-1 complex with Rab geranylgeranyl transferase and came to the conclusion that it functioned as a genuine intermediate of the prenylation reaction. Thus, we present the first example of the in vitro generation of a semisynthetic lipidated protein using the native chemical ligation method.
Alexandrov
Kirill
K
Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany. kirill.alexandrov@mpi-dortmund.mpg.de
Heinemann
Ines
I
Durek
Thomas
T
Sidorovitch
Vadim
V
Goody
Roger S
RS
Waldmann
Herbert
H
D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry; D017368:Protein Prenylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020691:rab GTP-Binding Proteins / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
D005456:Fluorescent Dyes; D009842:Oligopeptides; D000090802:rab7 GTP-Binding Proteins; D019883:Alkyl and Aryl Transferases; C075607:Rab geranylgeranyltransferase; D020691:rab GTP-Binding Proteins; D003545:Cysteine