Kalinin A, Thomä N H, Iakovenko A, Heinemann I, Rostkova E, Constantinescu A T, Alexandrov K
Max-Plank-Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Protein Expr Purif. 2001 Jun;22(1):84-91. doi: 10.1006/prep.2001.1423.
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.
哺乳动物II型香叶基香叶基转移酶（GGTase-II）是一种100 kDa的异二聚体，可催化两个20碳的香叶基香叶基从香叶基香叶基焦磷酸转移到Rab GTP酶的C端半胱氨酸残基上。这种修饰对于Rab蛋白的生物学活性至关重要。香叶基香叶基化可以使用重组GGTase-II在体外进行，但到目前为止，该酶的大规模生产具有挑战性。我们在此报告了一种双质粒表达系统的设计，该系统在大肠杆菌中产生的GGTase-II水平高达15 mg/L。该蛋白作为异二聚体产生，α亚基带有可切割的串联6His-谷胱甘肽S-转移酶（GST）标签，用于该酶的两步纯化。通过体外异戊二烯化和磷酸异戊二烯结合测定确定，纯化的酶具有功能活性。此外，带有GST标签的GGTase-II用于Rab7:REP-1复合物的制备性体外异戊二烯化。使用该方法，获得了10 mg双异戊二烯化的Rab7:REP-1复合物。
