SMPB-Modified Phosphatidylethanolamine Lipid Derivatives

Coupling via SMPB-Modified Phosphatidylethanolamine Lipid Derivatives 

Succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB) (Chapter 5, Section 1.6) is a heterobifunctional cross-linking agent that has an amine-reactive NHS ester on one end and a sulfhydryl-reactive maleimide group on the other. Conjugates formed using SMPB are linked by stable amide and thioether bonds. 

SMPB can be used to activate PE residues to contain sulfhydryl-reactive maleimide groups (Section 2). Lipid vesicles formed with MPB-PE components thus can couple proteins through available —SH groups, forming thioether linkages (Derksen and Scherphof, 1985) (Fig. 356). A comparison with SPDP-produced conjugates con- 

Prepare a liposome suspension, containing MPB—PE, at a total lipid concentration of 5 mg/ml in 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.2. Activation of DPPE with SMPB is described in Section 2. A suggested lipid composition for vesicle formation is PC cholesterol PG MPB—PE mixed at a molar ratio of 8 10 1 1. The presence of relatively high levels of cholesterol in the liposomal recipe dramatically enhances the conjugation efficiency of the component MPB—PE groups (Martin et al., 1990). Any method of emulsification to create liposomes of the desired size and morphology may be used (Section 1). 

Dissolve a sulfhydryl-containing protein at a concentration of at least 5 mg/ml in 0.05 M sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. The sulfhydryl groups on the protein molecule may be indigenous or created by any of the methods described in Chapter 1, Section 4.1. 

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