RESUMO
The high structural heterogeneity of smooth-type lipopolysaccharides (LPS) enormously complicates the isolation of their constituent molecular species. Proof of concept is given here on the feasibility of using preparative slab-PAGE to isolate highly homogeneous smooth-type LPS glycoforms. LPS species (from 3.6 to 14.2 kDa) from Escherichia coli K-235 were separated by preparative slab-PAGE and recovered by utilizing the combined on-gel LPS reverse staining, extrusion, and passive elution techniques. As a result, 15 electrophoretically pure LPS fractions were obtained. The LPS content in the recovered fractions ranged from 280 ng (intermediate mobility glycoforms) to 411 mug (highest mobility glycoforms). The quantities of LPS fractions were sufficient to allow quantitation of the Limulus amebocyte lysate (LAL) activities of these distinct-molecular-mass LPS species, in the range from (1.1 +/- 0.1)x10(3) to (8.7 +/- 0.3)x10(5) endotoxin units (EU)/mL, by standard LAL assay. We have thus definitively demonstrated that slab-PAGE may be a suitable platform to more selectively purify individual glycoform fractions from smooth-type LPS.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli/química , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/químicaRESUMO
High-pressure homogenization-extrusion (HPHE) is a method that can be used for downsizing large lipid vesicles with commercially available instrumentation (e.g., from Avestin Inc., Canada), which covers a full range of processing capacities from laboratory (0.5-3.5 mL) to large-scale continuous (1-1000 L/h) production. Consequently, the feasibility (at the laboratory scale) of using HPHE for producing DNA-loaded liposomes by the conventional dehydration-rehydration method was explored. HPHE-generated small unilamellar vesicles had a mean size in the range of 27-76 nm depending on the number of processing cycles and lipid (PC:DOPE:DOTAP or PC:DOPE:Ethyl-DOPC, 1:0.5:0.5, mol/mol) formulation. The size could be further regulated by the pore size (50 or 100 nm) of the extrusion membrane. Using plasmids for the V3 loop of HIV-1, and the capsid, E1 and E2 of hepatitis C, entrapment yields of 72-98.2% into dehydrated-rehydrated vesicles (DRV) were obtained over a wide range (0.309-2.5 mg) of DNA quantities. Most of the plasmid DNA was retained by liposomes even in the presence of sodium dodecyl sulfate (from 0.05% to 0.3%) and efficiently protected from nuclease-mediated degradation. Although the encapsulation process slightly decreased (in the range of 42.8-65.7%) the relative abundance of plasmid super coiled isoforms, the transfection efficiency of monkey kidney COS-7 cells with the plasmid DNA extracted from liposomes (9+/-0.4%) was similar to that of the non-treated DNA (8.7+/-0.2%), using the commercial SuperFect(R) Transfection Reagent. Also, it was found that an appreciable loss of lipid mass-either associated with the HPHE or the dehydration-rehydration steps-occurs during the liposome manufacturing process. These results at the bench scale are a useful reference for planning pilot or large-scale manufacture of DNA vaccine-containing liposomes.
Assuntos
Lipossomos , Vacinas de DNA/administração & dosagem , Animais , Células COS , DNA/metabolismo , Portadores de Fármacos , Lipídeos/análise , Tamanho da Partícula , Plasmídeos , Pressão , TransfecçãoRESUMO
We recently demonstrated that the combined use of lipopolysaccharide (LPS) reverse staining and high-efficiency passive elution techniques can be successfully used as a suitable interface between LPS slab-gel separation and electrospray ionization-mass spectrometry (ESI-MS) of LPS-derived oligosaccharides. Here, we extend our micropurification strategy for the analysis of O-deacylated LPS forms from Vibrio fischeri HMK after recovery from single reverse-stained LPS bands using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The quantities (30-40 microg) obtained from the two gel-resolved LPS bands were sufficient to allow MALDI-TOF-MS detection of O-deacylated LPS glycoforms at m/z 3767.1, 3890.1 for the high-molecular-weight or at m/z 2522.5, 2645.4, 2725.7, and 2848.7 for the low-molecular-weight LPS band. These LPS band heterogeneities resulted not only from variations in the oligosaccharide region of the LPS but also from two phosphorylation states of the lipid A (diphosphoryl and diphosphoryl plus a single phosphoethanolamine substitution). On the other hand, MALDI-TOF mass spectra of the separated LPS bands displayed reduced heterogeneity and increased signal-to-noise ratios as compared to spectra of the unpurified LPS. Furthermore, micropurification of LPS bands prior MALDI-TOF-MS led to a higher sensitivity of detection of less abundant low-molecular-weight LPS glycoforms. Taken together, this and our previous study on gel-micropurified LPS using ESI definitively show how one can unambiguously determine the different molecular species contained within each gel-separated LPS band, their relative abundance and oligosaccharide sequences.