Intein-Mediated Synthesis of Geranylgeranylated Rab7 Protein in Vitro

Abstract

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.

Diels–Alder Ligation of Peptides and Proteins

“…Therefore, differently and site-specific labeled Rab proteins are considered versatile probes for the study of such biological events. [110][111][112][113][114] Rab7 (207 AA): MTSRKKVLLKVIILGDSGVGKTSLMNQYVNKKFSNQYKATIGADFLTKEVMVDDRLVTMQ IWDTAGQERFQSLGVAFYRGADCCVLVFDVTAPNTFKTLDSWRDEFLIQASPRDPENFPF VVLGNKIDLENRQVATKRAQAWCYSKNNIPYFETSAKEAINVEQAFQTIARNALKQETEV ELYNEFPEPIKLDKNERAKASAESCSC Figure 32. Structural details for Rab7 protein complexed with GNP and magnesium ion.…”

“…[110] The method uses a genetically engineered intein and a chitin binding domain (CBD) as fusion partners to express and purify the desired Rab thioester (Section 2.6.1) and has been successfully applied for the semi-synthesis of fluorescent labeled and lipidated Rab proteins by the groups of Dr. Kirill Alexandrov and Prof. Dr. Herbert Waldmann. [110][111][112][113][114] For the dienyl linker construction, three different cysteinyl hexadiene linkers 57, 58…”

“…The reductively cleavable StBu group was chosen as the protecting group of the N-terminal cysteine to prevent oxidative reactions. [111] The deproctection of the cysteine is carried out in situ during protein ligation by using an excess of thiol agent. The synthesis of the linker 57 began with the assembly of Fmoc-protected compound 60 on solid phase using 2-chloro-trityl resin, followed by esterification of the resulting carboxylic peptide 60 with hexadienol 1 and final removal of the Fmoc protecting group (Section 4.1.3).…”

“…For the construction of the C-terminal hexadienyl Rab7 protein, recombinant truncated Rab7 thioester (Rab7∆C3-thioester) was ligated with peptide hexadienyl ester under reducing conditions (MESNA) overnight at 16°C (Scheme 27). [111] An excess of 20 equivalents of the dienyl compound was used to ensure complete conversion. The ligation was carried out in the presence of guanosine 5´-diphosphate (GDP) and magnesium chloride to assure protein stabilization.…”

“…[111][112][113] The purification of the Rab protein 64 ( Figure 37) was performed following the procedure described for the isolation of semisynthetic prenylated Rab proteins. [111] After incubation of the Rab hexadienyl ester 63 with the maleimide, the resulting protein precipitate was separated by centrifugation and washed with methanol in order to remove the excess of unligated dienophile. The resulting pellet was dissolved in buffer containing 6M guanidinium chloride and DTT and then refolded by a 25-fold dilution with renaturation buffer.…”