To prepare b-galactosidase-bound onto Ni 2? -NTA liposomes (further referred to as AnExIL-ON), b-galactosidase was expressed in bulk and subsequently bound onto liposomal bilayers as described below. Ninety-ll of IVTT and pIVEX2.2EM-LacZ as a pDNA template with a final concentration of 20 nM was used for bulk expression of a b-galactosidase with a C-terminal 6HIS-tag. IVTT mix with DNA template was incubated at 30°C for 3 h. After the expression was completed, 75 ll of the reaction mix containing expressed b-galactosidase was added to the NTA-lipid cakes (6 lM lipids) and incubated for 10 min at room temperature to form liposomes. The expressed b-galactosidase has a 6-histidine residue which binds to Ni 2? ions of Ni 2? -NTA liposomes. To remove the non attached b-galactosidase, the liposomes were washed three times with 0.5-1.5 ml phosphate buffer saline (PBS, NaCl concentration was adjusted to make it isotonic with the IVTT mix) by centrifugation at 8,000-9,0009g for 10 min at 4°C. After washing, the liposomes were re-suspended in 50 ll PBS.