Expression of mammalian geranylgeranyltransferase type-II in Escherichia coli and its application for in vitro prenylation of Rab proteins.
Mammalian geranylgeranyltransferase type II (GGTase-II) is a 100-kDa heterodimer that catalyzes the transfer of two 20-carbon geranylgeranyl groups from geranylgeranyl pyrophosphate onto C-terminal cysteine residues of Rab GTPases. This modification is essential for the biological activity of Rab proteins. Geranylgeranylation can be performed in vitro using recombinant GGTase-II but so far large-scale production of the enzyme was challenging. We report here the design of a two plasmid expression system that will produce GGTase-II at levels as high as 15 mg/L in Escherichia coli. The protein was produced as a heterodimer with the alpha subunit bearing a cleavable tandem 6His-glutathione S-transferase (GST) tag that was used for two-step purification of the enzyme. Purified enzyme was functionally active as determined by in vitro prenylation and phosphoisoprenoid binding assay. Furthermore, the GST-tagged GGTase-II was used for preparative in vitro prenylation of the Rab7:REP-1 complex. Using this procedure, 10 mg of doubly prenylated Rab7:REP-1 complex were obtained.
Kalinin
A
A
Max-Plank-Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Thomä
N H
NH
Iakovenko
A
A
Heinemann
I
I
Rostkova
E
E
Constantinescu
A T
AT
Alexandrov
K
K
D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008322:Mammals; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017368:Protein Prenylation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000090802:rab7 GTP-Binding Proteins
D011994:Recombinant Proteins; D000090802:rab7 GTP-Binding Proteins; D019883:Alkyl and Aryl Transferases; D020691:rab GTP-Binding Proteins