*4.1. Protein Preparation*

The gene coding for eIF4E initiation factor from the lettuce (Lactuca sativa cultivar Salinas GenBank AF 530162) and its derived molecular species were cloned into the vector pENTR/D-TOPO® (Invitrogen, Carlsbad, CA, USA). They were transferred into pDESTTM17 using the Gateway® recombinant Technology to allow production of N-terminal fusions with an hexahistidine tag (Invitrogen). In addition, full length eIF4E and eIF4E (Δ1–46) were cloned into the vector pENTR/SD/D-TOPO® (Invitrogen) and transferred into the Gateway pDESTTM14 expression vector according to manufacturer's instructions to obtain untagged full length eIF4E and eIF4E(<sup>Δ</sup>1–46). The constructs were introduced into *E. coli* (BL21-AI strain), expression and purification of the His-tagged proteins were performed on ion metal affinity chromatography followed by m7GTP-Sepharose 4B (GE Healthcare, Amersham, UK) as previously described [39]. Untagged proteins were obtained by one step affinity purification on m7GTP sepharose 4B. The Lettuce mosaic virus VPg (isolate AF199GenBank AJ2 78854) coding sequence was cloned into the pTrcHis C expression vector downstream from an hexahistidine tag (Invitrogen). The vector contains a specific enterokinase cleavage site in frame with the protein for proteolytic tag removal. The protein was produced and purified as previously reported [39] except that the final monoQ chromatographic step was replaced by a size exclusion chromatography on a Superdex 75 HR 10/30 column (GE Healthcare) in 20 mM Hepes pH 8, 300 mM NaCl, and 2 mM DTT. For His-tag removal, this last step was omitted and the protein was diluted twice in the same buffer with reduced ionic strength (150 mM NaCl) containing 1 mM CaCl2, 0.1% Tween-20. His tagged enterokinase (0.02 mg for 0.2 mg VPg) was added and the protein mix was dialyzed overnight at 4 ◦C against the same buffer. The protease and uncleaved tagged VPg were subsequently trapped on a NiNTA resin and the pure free VPg form was recovered in the flow through (30–40% yield).

Protein labelling with *N*-(iodoacetyl)-*N* -(5-sulfo-1-naphtyl)ethylenediamine, (IAEDANS).

Cysteine residues were reduced before labelling. The VPg or eIF4E solutions (0.2 mg/mL) were dialyzed overnight in 25 mM HEPES, pH 7.5, 6 mM βMe, 2 mM AcNa, 5 mM EDTA, 25 mM DTT, NaCl 0.3 M at 4 ◦C. The protein solutions (1 mL) were loaded on a 5 mL G25 column equilibrated in the coupling buffer (25 mM HEPES, pH 7.5, NaCl 0.3 M). A 10 times molar excess of IAEDANS was added in the same buffer and the mix was incubated in the dark at 25 ◦C for 2 h. The reaction was stopped by addition of DTT (25 mM final concentration). A buffer exchange was performed over a G25 equilibrated in 25 mM HEPES, pH 7.5, 150 mM NaCl. About 1 and 2 AEDANS molecules were bound per VPg and eIF4E respectively.
