FSL-RFG(Maleimide) FSL construction kit

Abstract

The FSL-RFG(Maleimide) FSL Construction Kit is for use in creating Function-Spacer-Lipid (FSL) constructs for use in non-covalent cell-surface modification/engineering of cellular membranes, viral particles, liposomes, or other surfaces [1-10]. FSL-RFG(Mal) is one of several FSL constructs with Reactive Functional Groups (RFG); with this construct having maleimide as its Function group. The semi-rigid Spacer in this molecule is constructed via modified hexapeptide unit (Gly-Gly-Ida)2 coupling to both amino groups of ethylenediamine and has been designed to ensure accessibility for target binding/external interactions and proper presentation of functional peptides at a cell or virion surface as well as imparting good solubility to the construct. Electrostatic repulsion forces of spacer’s anionic groups probably favor uniform distribution of the incorporated constructs on the membrane surface [11]. The diacyl phospholipid derived from unsaturated fatty acids is a prerequisite for spontaneous incorporation into cell membranes. This FSL-RFG(Maleimide) FSL Construction Kit cat # 960819-1-R&D (includes a detailed procedure and contains reagents sufficient for one FSL preparation on a milligram scale from cysteine-containing peptides (Figure 1), proteins or any other thiols of biological interest. The effective synthetic approach is based on the well-known Michael nucleophilic addition to maleimides, which react fast and selectively with SH-groups in the pH range 6.5-7.5 producing stable thioether linkages completely stable at physiological conditions [12-15]. The reaction half-life between millimolar concentrations of maleimide and thiol is estimated to be of the order of few seconds [14,15]; but more complex and heavy molecules of biochemical interest interact somewhat slower even when applied in 10-fold excess and durations of at least 2 hours are recommended [16]. The protocol described here is optimized for this kit using FSL-RFG(Mal) with generic peptides and addresses problems which may be encountered if purification of completed FSL constructs is required.