Liposomes proteins using

Native chemical ligation also can be extended to the conjugation of peptides or proteins to other molecules or surfaces. For instance, Reulen et al. (2007) prepared liposomes that contained cysteine-PEG-phospholipid derivatives and then coupled thioester-modified peptides or proteins to form a protein-liposome conjugate. Using this procedure, approximately 100 molecules of a collagen binding protein could be coupled to the cysteine-containing liposomes. 

Bz s-imidoesters like DMS may be used to couple proteins to PE-containing liposomes by crosslinking with the amines on both molecules (Figure 22.24). However, single-step crosslinking procedures using homobifunctional reagents are particularly subject to uncontrollable polymerization of protein in solution. Polymerization is possible because the procedure is done with the liposomes, protein, and crosslinker all in solution at the same time. 

Reulen, S.W.A., Brusselaars, W.W.T., Langereis, S., Mulder, W.J.M., Breurken, M., and Merkx, M. (2007) Protein-liposome conjugates using cysteine-lipids and native chemical ligation. Bioconjugate Chem. 18(2), 590-596. 

DNA and/or protein vaccine entrapment in DRV liposomes is monitored by measuring the vaccine in the suspended pellet and combined supernatants. The most convenient way to monitor DNA entrapment is by using radio-labelled or DNA. For protein entrapment, the use of I-labelled protein tracer is recommended. If a radiolabel is not available or cannot be used, appropriate quantitative techniques should be employed. To determine DNA or protein by such techniques, a sample of the liposome suspension is mixed with Triton X-100 (up to 5% final concentration) or, preferably, with isopropanol (1 1 volume ratio) so as to liberate the entrapped materials. However, if Triton X-100 or the solubilized liposomal lipids interfere with the assay of the materials, liposomal lipids or the DNA must be extracted using appropriate techniques (6). Entrapment values for protein and DNA, whether alone or coentrapped, range between about 20% to 80% (protein) and 30%i to 100%i (DNA) of the initial material depending on the DNA or protein used and, in the case of DNA, the presence or absence of cationic charge. Values are highest for DNA when it is entrapped into cationic DRV (typical values in Table 1). 

Conventional liposomes and lipid complexes. Liposomes were used initially as a model system for cellular membranes to study the biochemistry of membrane proteins.85 Consequently, when liposomes were first tried as a drug delivery system, their bilayers were composed of un-derivatized naturally occurring lipids. Most of such conventional liposomes are taken up by the MPS phagocytes within a few hours of injection, mostly by liver Kupffer cells and spleen macrophages.9 Inside the endosomes and lysosomes of those cells, liposomes are degraded. If the liposomal drugs are membrane permeable, they then can diffuse from the endosomal compartments to the cytoplasm of the macrophage cells and slowly reenter the blood circulation. Because such a clearance... 

The most widely used procedure for generating protein-liposome conjugates involves modification of the protein with SPDP, followed by deprotection with DTT and conjugation to SMPB-derivatized liposomes. We use the following protocol to generate such conjugates using IgG (see Note 2). 

Modification with NGPE allows binding of several hundred protein molecules per 250 nm diameter liposome (44). Using 125I-labeled antibody, the efficacy of protein binding varied between 65 and 75% and did not depend on the presence of PEG-PE in the lipid mixture. The unbound antibody is separated on a Bio-Gel A15M column. Liposomes obtained are serially filtered through polycarbonate filters with pore sizes of 0.6,0.4, and 0.2 pm. The actual size (normally 160-190 nm) and distribution of the liposomes are determined with a particle size analyzer. 

However, the study of hydrophobic proteins using Biacore systems is possible using protocols specially designed for the purpose. For example, proteins may be firstly incorporated into liposomes and then immobilized on hydrophobic sensor surfaces (Sect. 3.3). It has been reported that hpid bilayers have been tethered to a sensor surface via hydrophihc spacers immobilized on a plain gold chip into which membrane-spanning proteins are then inserted [57]. The emphasis of this technique rests on encasing sensitive protein domains within a hpid microenvironment in which they can assume a native, functional structure. 

It is possible to incorporate membrane proteins in liposomes without using detergents (or in the presence of low detergent concentrations). In particular, you can let membrane proteins move directly from the native membrane into liposomes, without inserting a solubilization step. This technique usually requires specialized and highly purified phospholipids and a precise knowledge of their properties. 

Jung, K., Kim, T.W., Park, H.G., and Sob, H.T. 2010. Specific clorimetric detection of proteins using bidentate aptamer-conjugated polydiacetylene (PDA) liposomes. Adv. Funct. Mater. 20 3092-3097. 

The total amount of proteins transferred was relatively larger in suspension culture than that in monolayer culture (Table 5). Even after protein transfer, particle size of liposomes almost unchanged and efficiency of recovery of lipids was rather good. Viability of the cell after the exposure to liposome was largely affected by the method of culture. In the suspension culture, the cell viability was less than 20 % after the exposure. In the monolayer culture system, however, the viability of the cell was maintained more than 60 %. When simple DMPC liposome was used, the viability was almost 80 %. 

For the monolayer culture, major proteins transferred were 95, 77, 68, 48 (MSH receptor ), and 35 kD, while they were 112 (vitronectin receptor or adhesion protein ), 101, and 51 kD for the suspension culture (Figure 4). When DMPC(20)/DDPC(80) liposome was used in the suspension culture, in addition, a tumor antigen ganglio-side, CM3, was transferred accompanied by proteins. 

Fleiner M, Benzinger P, Fichert T et al (2001) Studies on protein-liposome coupling using novel thiol-reactive coupling lipids influence of spacer length and polarity. Bioconjug Chem 12 470 75... 

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