Post release modification of viral envelopes

Abstract

Disclosed are methods of treatment of a subject, such as a method of vaccination, immunomodulation or gene therapy of a subject. These methods comprise administering to the subject a modified enveloped viral particle, wherein the modified enveloped viral particle has been obtained by a method comprising the steps of a) incubating a fluid containing enveloped viral particles with one or more reactants consisting of a hydrophilic target domain and a lipophilic membrane anchor domain, wherein the lipophilic membrane anchor domain becomes integrated into the lipid double layer of the envelope of the viral particle, wherein the hydrophilic target domain becomes exposed to the fluid; and b) separating enveloped modified viral particles from excessive reactants.

Claims

1. A method of stimulating an immune response or immunomodulation of a subject, the method comprising: a) incubating a fluid containing enveloped viral particles with one or more reactants consisting of a hydrophilic target domain and a lipophilic membrane anchor domain, wherein the lipophilic membrane anchor domain becomes integrated into the lipid double layer of the envelope of the viral particle, wherein the hydrophilic target domain becomes exposed to the fluid, wherein the hydrophilic target domain is selected from the group consisting of polysaccharides, nucleic acids, dyes, radioactive ligands, fluorescent dyes, synthetic beads, magnetic particles and proteins or polypeptides comprising a protein tag; b) separating enveloped modified viral particles from excessive reactants; and c) administering to the subject the modified enveloped viral particle.

2. The method according to claim 1, wherein the viral particle is selected from the group consisting of a wild-type virus, an attenuated virus, an empty virus particle and a genetically modified viral vector.

3. The method according to claim 1, wherein the viral envelope has a protein to lipid ratio between 50:50 and 90:10 mol %.

4. The method according to claim 1, wherein the lipophilic membrane anchor domain is selected from the group consisting of phospholipid-polyethyleneglycol, stearyl, palmityl, myristyl, cholesterol, chelator lipid nitrilotriacetic acid ditetradecylamine (NTADTDA) and glycosylphosphatidylinositol (GPI).

5. The method of claim 1, wherein the protein is an immuno-stimulatory protein.

6. The method according to claim 1, wherein the protein or the polypeptide is an enzyme, an antibody, a receptor, a marker protein, a fluorescence protein, a complement inhibitor or a cytokine.

7. The method according to claim 1, wherein the enveloped viral particle is selected from the group consisting of Arenaviridae, Bunyaviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae, Poxviridae, Retroviridae, Rhabdoviridae and Togaviridae.

8. The method according to claim 1, wherein the enveloped viral particle is one of a retrovirus, a poxvirus, a herpesvirus, an influenza virus and a lentivirus.

9. The method according to claim 8, wherein the enveloped viral particle is one of a mouse leukemia virus, a feline herpesvirus and a vaccinia virus.

10. The method according to claim 1, wherein the enveloped viral particle comprises a genetically modified genome compared to its wild-type form.

11. The method of claim 1, comprising prior to step a) the step of obtaining enveloped viral particles from a suspension fluid.

12. A method of treating a subject, the method comprising: a) incubating a fluid containing enveloped viral particles with one or more reactants consisting of a hydrophilic target domain and a lipophilic membrane anchor domain, wherein the lipophilic membrane anchor domain becomes integrated into the lipid double layer of the envelope of the viral particle, wherein the hydrophilic target domain becomes exposed to the suspension fluid, wherein the hydrophilic target domain is selected from the group consisting of polysaccharides, nucleic acids, dyes, radioactive ligands, fluorescent dyes, synthetic beads, magnetic particles and proteins or polypeptides comprising a protein tag; b) separating enveloped modified viral particles from excessive reactants; and c) administering to the subject the modified enveloped viral particle.

13. The method according to claim 12, wherein the viral particle is selected from the group consisting of a wild-type virus, an attenuated virus, an empty virus particle and a genetically modified viral vector.

14. The method of claim 12, wherein the treatment is selected from the group consisting of gene-therapy, vaccination and immunomodulation.

15. The method of claim 12, comprising prior to step a) the step of obtaining enveloped viral particles from a suspension fluid.

16. A method of modifying at least one enveloped viral particle and detecting the modified enveloped viral particle, the method comprising: a) incubating a fluid containing enveloped viral particles with one or more reactants consisting of a hydrophilic target domain and a lipophilic membrane anchor domain, wherein the lipophilic membrane anchor domain becomes integrated into the lipid double layer of the envelope of the viral particle, wherein the hydrophilic target domain becomes exposed to the fluid, wherein the hydrophilic target domain is selected from the group consisting of polysaccharides, nucleic acids, dyes, radioactive ligands, fluorescent dyes, synthetic beads, magnetic particles and proteins or polypeptides comprising a protein tag; b) separating enveloped modified viral particles from excessive reactants; c) contacting one of a cell, a tissue and a subject with a modified enveloped viral particle; and d) detecting the modified enveloped viral particle bound to the cell, tissue or subject.

Description

SHORT DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A depicts the painting of retroviral and lentiviral vector particles in a schematic overview. This overview is based on the following experiment: concentrated supernatants from retroviral (RV) or lentiviral (LV) producer cell lines (293gpalfpLXSNeGFP and STAR-A-HV, respectively) are incubated with purified and concentrated CD59his for 3-20 hours at 37 C. under constant shaking. After incubation samples are purified by ultracentrifugation (2 hrs, 20 000 rpm, 4 C.) to remove non-virus-associated proteins. Before analysis of CD59his, endogenous CD59 is removed by using magnetic nickel beads (Promega). Samples were analysed by immunoblotting using antibodies directed specifically against CD59, MLV capsid (CA) and HIV-1 p24. FIG. 1B shows the analysis of painted retrovirus: concentrated supernatants from parental cells (PC) and virus producing cells (VP) were incubated in the presence or absence of CD59his for 21 hours at 37 C. under constant shaking. In addition cell culture medium (ME) was also incubated under the same conditions. After purification as detailed above, cells were analysed by immunoblotting. Results show that CD59his is only retained during purification in the presence of virus and CD59his (RV and LV, upper panels respectively, compare lanes VP and VP+) indicating association of the protein with viral particles. On the same gels, either 5 or 10% respectively of the amount of CD59his used for viral painting was loaded to assess efficiency of painting. Levels of viral gag proteins are shown via immunoblots using MLV capsid and HIV-1 p24 antibodies. Viral proteins are only present in supernatants derived from viral producers. (RV and LV, lower panels respectively, compare lanes VP and +). FIG. 1C shows the specificity of viral painting: concentrated viral supernatant were mixed with CD59his and the same amount of a non-GPI protein (rat IgG; MW 150 kD; Dako). The sample was incubated for approximately 20 hours. Aliquots are taken before and after ultracentrifugation and silver-stained to assess protein content. 100% and 10% of the used amount of IgG were loaded for comparison. Ultracentrifugation removes the majority of proteins as well as the IgG contaminant. FIG. 1D shows infectivity after Painting: Virus supernatants post-painting are purified by ultracentrifugation and used to infect target HeLa cells. After 36 hours supernatant is removed and analysed by immunoblotting for presence of CD59his to confirm painting. Cells analysed by flow cytometry. No infection was observed in HeLa cells treated with medium (mock) or supernatant from parental cells that was incubated with CD59his (PC+). Virus supernatant in the absence or presence of CD59his (VP; VP+) shows infection rates of approximately 4%. Supernatants treated with CD59his performed slightly worse in infection experiments. As infection control viral supernatant saved before painting (one tenth of the volume used for infection after painting) was used (VP). Infectivity was reduced significantly after painting. Error bars represent means+/standard deviation.

(2) FIG. 2A schematically shows that the-N-terminal signal peptide (SP) of the nascent polypeptide chain leads to translocation to the ER. The transamidase complex located at the ER membrane, recognizes the Glycosylphosphatidylinositol (GPI) anchoring signal sequence (GSS) and replaces it with the preformed GPI anchor. FIG. 2B schematically shows that a common backbone structure is observed in GPI anchors. Linkage to the protein part is achieved by an amide bond to phosphoethanolamine. The following carbohydrate core consists of three mannose and a glucosamine residue. The phosphoinositol group links the hydrophobic lipid part, mainly aryl, acyl or ceramide type lipids, to the remaining GPI anchor. The hydrophobic moieties are inserted into the outer leaflet of the cell membrane. Sites of cleavage for mammalian GPI-specific phospholipases C and D (GPI-PLC and GPI-PLD, respectively) are indicated by arrows.

(3) FIG. 3: Serum protection after viral painting. An experiment (n=1) was carried out to determine serum protection of virions painted with CD59his. After painting incubation and post-painting purification steps painted viral particles were challenged with human serum for 1 hour at 27 C., as were unpainted viral particles. 48 hours post infection the target HeLa cells (ATCC No. CCL-2) were analysed for infection by flow cytometry. Infectivity of unpainted viral particles was reduced to about 16% after challenging with human serum, compared to not challenged viral particles. The observed infectivity loss in painted viral particles was less pronounced (to 60%), as expected if serum protection from CD59his takes place.

(4) FIG. 4: Varying amounts of GPI-anchored proteins and viral particles. Varying amounts of CD59his (1, 4, 16 relative amounts) were incubated with the same amount of viral particles (2 relative amount). After post-painting purification steps immunoblots for CD59 show more association of CD59his to viral proteins when higher concentrations of CD59his were used as starting material. When the same concentration of CD59his was used to paint different amounts of viral particles (1, 2, 4 relative amounts) a medium concentration of viral particles seemed to perform best (2 relative amount).

(5) FIG. 5: Painting of feline herpesvirus 1 (FHV-I):

(6) Concentrated supernatants from CrFK cells infected with FHV-I or treated with hygromycin were used in a painting experiment, along with cell culture mediumincubation was carried out in the presence or absence of CD59his for 20 hours before post-painting purification by ultracentrifugation. Immunoblot using specific antibodies directed against CD59 were carried out. Signals can be detected for samples incubated in the presence of CD59 from supernatants from infected (CrFKFHV+) andto a lesser degreehygromycin-treated cells (lanes CrFKHyg+). To determine activity of virus post-painting, aliquots of samples were used to infect CrFK cells. The cell destruction or cytopathic effect (CPE) was analysed 24 hours post infection by phase contrast light microscopy. Strong CPE was detected in samples containing FHV-I particles only (CrFKFHV and +, respectively). This indicates that virus remains infectious during the procedure. Cytopathic effects (CPE) upon infection of CrFK cells with painted FHV-I particles. In the presence of complete, biologically active virus CrFK cells are infected and damaged (see lane FHV). After painting CPE can be detected in cells infected with the CrFKFH V samples only, confirming presence of active virus post-painting (see lanes CrFKFHV and +). A confluent monolayer is observed in samples not containing viral particles (Medium/+; CrFK/+).

(7) FIG. 6: Painting with GPI-anchored green fluorescent protein.

(8) Lentiviral particles derived from STAR producer cells were incubated with GPI-anchored monomeric green fluorescent protein (mGFP-GPI). Post-painting samples were purified by ultracentrifugation and analysed by immunoblotting for presence of mGFP-GPI (upper panel) and p24 (lower panel). mGFP-GPI was only present when viral particles were present, and the sample had been treated by painting with mGFP-GPI (lane VP+) and to a lesser extent when supernatant from non-virus producing parental cells was treated by painting (lane PC+) due to the presence of lipid vesicles of non-viral origin.

(9) FIG. 7: Magnetic nanoparticles (MNP) associate specifically with recombinant GPI proteins GPI-anchored green fluorescent protein was mixed with magnetic nanoparticles (Nickel-NTA-coated iron core particles, average size 5-10 nm). MNPs associated with the target proteins could be retained during three washing procedures after magnetic manipulation (lane B). Most of cellular protein was lost (compare lanes B and A, bottom panel, Coomassie-stained gel), whereas a comparatively large proportion of the target protein stayed associated throughout the process (compare lanes B and A, top panel, immunoblot for GFP). This indicates that magnetic manipulation of GPI-anchored proteins is possible when using magnetic nanoparticles. Lane M exhibits the molecular weight marker.

EXAMPLES

Example 1: Production of CD59his

(10) CrFKCD59hisneo cells expressing the recombinant CD59his were derived from parental CrFK cells by lipofection using lipofectin reagent according to manufacturer's instructions (Invitrogen) with pCD59hisneo. For generation of pCD59hisneo a PCR fragment derived from cDNA (using primers CD59(2)FKHindIII 5-cacgacaagcttaccatgggaatccaaggaggg tctgtcctgtt-3 SEQ ID No: 5) and CD59(2)RApaI5-atgacgggcccttagggatgaaggctccaggctgctgccagaa-3 SEQ ID No: 6) from HEK293 cells was cloned into the expression vector pcDNA3 (Invitrogen). The his-tag was introduced by a two-step mutagenesis PCR protocol, using first two primer pairs (CD59(2)FKHindIII & CD59RHis 5-gtgatggtgatggtgatggctatgacctgaatggcagaag-3 SEQ ID No: 8; CD59FHis 5-catcaccatcaccatcacctgcagtgctacaactgtccta-3 SEQ ID No: 7 and CD59(2)RApaI) in two different PCR reactions. Subsequently a mix of both primary fragments was hybridized and amplified using primers CD59(2)FKHindIII and CD59(2)RApaI. The fragment was recloned into pCDNA3 using the HindIII and Apal sites. 293gpalfpLXSNeGFP are derived from HEK293 cells (Ikeda (2003); Klein (1997); Pambalk (2002)). STAR-A-HV (Wilhelm (2007)) are derived from HEK293T cells).

Example 2: Purification of CD59his

(11) 4-6 confluent T175 flasks of CrFKCD59hisneo were harvested by scraping after washing cells with 10 ml PBS. Cells were scraped into a total of 25 ml sample application buffer (50 mM TrisHCl, 50 mM NaCl, 35 mM Imidazole, 0.5% sodium deoxycholate, 1% NP40, pH 7.4). 80 l of protease inhibitor complex (Sigma) was added before sonification of samples for 30 seconds. Samples were incubated for 30 minutes on ice before centrifugation for 30 minutes at 2000 g. Samples were filtered through 0.2 m filters (Sarstedt) before application to a ktaPrime plus FPLC device (GE Healthcare). Prepacked 5 ml HisTrap FF Crude columns (GE HealthCare) were used. Samples were washed using washing buffer (50 mM TrisHCl, 50 mM NaCl, 35 mM Imidazole, pH 7.4) and eluted from columns by elution buffer (50 mM TrisHCl, 50 mM NaCl, 600 mM Imidazole, pH 7.4). Fractions were collected during elution. Presence of CD59his in fractions was determined by immunoblotting. Positive fractions were pooled and concentrated by ultrafiltration using Amicon Ultra filter devices (Millipore, 5 kD molecular weight cut-off). Samples were washed twice with 5 ml painting buffer (50 mM TrisHCl, 50 mM NaCl, pH 7.4). Concentrations were measured using the DC protein assay (BioRad).

Example 3: Painting of Virus with CD59his

(12) Supernatants from the stable lentiviral producer cell line STAR-A-HV (14) or the MLV-based retroviral producer cell line 293gpalfpLXSNeGFP (15, 16, 17) were harvested, filtrated through 0.45 m filters (Sarstedt) and viral particles were concentrated by ultracentrifugation (2 hrs, 20 000 rpm, 4 C.) in a Beckmann XL-70 ultracentrifuge using a SW28 rotor and resuspended in DMEM cell culture medium (Gibco), before incubation with CD59his at final concentrations between 20 and 100 ng/l for 21-24 hours at 37 C. and 5% CO.sub.2. For painting, supernatants derived from concentration of 2-6 T175 culture flasks were incubated with purified protein at final concentrations between 20 and 100 ng/l or painting buffer alone. Incubation was carried out at 37 C., 5% CO2 under constant shaking. Incubation times were 3 (infection experiments) to approximately 21 hours (standard experiments). To separate potentially painted virus from free GPI-linked proteins, samples were diluted by addition of 34 ml of DMEM and ultra-centrifuged (2 hrs, 20 000 rpm, 4 C.). To allow for the differentiation between recombinant CD59his and endogenous CD59 present on virus producer cells, samples were subjected to purification with Ni-magnetic particles (MagneHis kit, Promega) after painting according to the instructions of the supplier. and ultracentrifugation to remove endogenous CD59 derived from producer cells (for an overview of procedure see FIG. 1A). CD59his was detected only in samples containing virus and purified CD59his, suggesting that both constituents are necessary (FIG. 1B, lane VP+). No influence on painting was observed as a result of the used media (FIG. 1B, lane ME) or the parental (non-virus producing) cells (FIG. 1B, lane PC+) used. However, if cells undergo considerable stress i.e. overgrowing in culture prominent amounts of non-viral membrane vesicles can be shed (18) leading to the potential for painting of these exosomal bodies as well. In addition, post-painting procedures were sufficient to remove unpainted CD59his (seen by the absence of a signal for CD59his in the ME+ sample, FIG. 1B) as well as endogenous CD59 (seen by the absence of a signal for CD59his in the PC sample, FIG. 1B).

(13) Proteins may however stick to viral envelopes regardless of GPI-anchoring in a non-specific manner. Silver staining of painted samples before and after purification via ultracentrifugation showed that the majority of proteins are removed in the purification step (FIG. 1C). In addition, we added rat IgG at the same levels as CD59his to the painting reaction. IgG was not retained by the virus as the CD59his was (FIG. 1C). This indicates that the process is at least semi-specific. Potentially proteins with pronounced hydrophobic stretches e.g. trans-membrane proteins could interact in a way similar to GPI proteins with lipid membranes.

(14) Optimisation was carried out to determine the minimal incubation time necessary for membrane re-insertion. Preliminary results suggested that an incubation time of 3 hours is sufficient for maximal viral painting. Using the minimal incubation time, painting experiments were repeated, to assess infectivity of painted virus. HeLa (ATCC No. CCL-2) cells are infected with painted virions and analysed by flow cytometry 36 hours post infection. Supernatant after infection was collected and analysed for CD59his to confirm painting. (FIG. 2). Painted virus remains infectious, however at reduced levels. Differences in infectivity between samples that received CD59his and mock-painted samples that did not receive GPI proteins are small (FIG. 2, compare samples HV and HV+). The difference of infectivity to samples before painting was comparatively large (approximately 15-fold, FIG. 2, compare samples HV, HV and HV+). This indicates that the reduction in infectivity is rather a result of the duration of the process than the process itself.

Example 4: Painting Stoichiometry

(15) Calculations of the stoichiometry of the viral painting process, especially the numbers of GPI proteins incorporated per virus are based on viral titers determined by product enhanced reverse transcriptase (PERT) and by determination of viral painting efficacy from immunoblots. The density of CD59 per virion is defined as the number of total associated molecules N.sub.MA divided by the number of virions N.sub.V, determined by product enhanced reverse transcriptase (PERT) assay. The PERT assay was carried out as described in (19). Before electroblotting (1.1 mA/cm.sup.2) onto PVDF membranes (Hybond P, GE HealthCare). samples were electrophoretically separated on pre-cast 4-12% gradient gels (NuPage, Invitrogen). Monoclonal antiCD59 was purchased from Serotec. Mouse anti human HIV-1 p24 was purchased from Polymun Scientific (Vienna). MLV anti capsid antibody was purified by Biomedica. HRP-conjugated anti-rat and anti-mouse secondary antibodies were purchased from DakoCytomation. Signal detection was carried out using the ECLplus kit (GE HealthCare)

(16) The density (D) of CD59his molecules per virion is dependent on the amount of CD59his (M [g]), the efficacy of the association process (E.sub.A) and the number of virions (N.sub.v), determined by product enhanced reverse transcriptase (PERT) assay. The constant factor k contains the parameters supposed to not change between experiments, such as the molecular weight (M.sub.w) of the GPI protein (20 kDa), the efficacy of purification (E.sub.p) and the Avogadro number (N.sub.a). Following formula can be used for calculation of the stoichiometry:
k=(E.sub.pN.sub.a)/(M.sub.w10E9);D=k(ME.sub.A)/N.sub.v

(17) Results for the experiments depicted in FIG. 1B suggested that between 5 and 250 molecules can be found per virus. Experiments carried out using either the same concentration of CD59his on varying viral concentrations or vice versa showed that the amount of incorporation of CD59his into viral envelopes is dependent on viral titers and CD59his concentration (see FIG. 4), whereas the number of inserted CD59his molecules increase with increased amounts of CD59his. In parallel the infectivity of the viral particles decrease with increased numbers of inserted CD59his due to a steric hindrance of natural viral envelope proteins. The best relation between the number inserted compounds and infectivity can be achieved with 50 to 150 CD59his molecules per virion.

Example 5: Infection of HeLa Cells and Flow Cytometry

(18) For infection 8-910.sup.5 HeLa target cells (ATCC No. CCL-2) were seeded 6 hours prior to infection in 6 well plates. Virus supernatants after post-painting ultracentrifugation were diluted to 1 ml with DMEM supplemented with 10% FCS (Gibco) and 10 l/ml polybrene (0.8 g/l). After 36 hours Supernatants were saved for analysis of CD59his content. Cells were trypsinised, fixed, washed 2 times in PBS and analysed for expression of eGFP in a FACsCalibur flow cytometer (BectonDickinson) using CellQuest software.

Example 6: Painting of Feline Herpesvirus 1 (FHV-I)

(19) Crandell feline kidney cells (CrFK, ATCC No. CCL-94) were infected with FHV-I (2 ml concentrated suspension per T 175 flask) and incubated until complete destruction of cells took place (approximately 48 hours). In parallel, the same amount of CrFK cells was treated with hygromycin (Invitrogen, 200 g/ml final concentration) to simulate the cell damage usually associated with FHV infection. The supernatants were harvested by ultracentrifugation (2 hrs, 2OK rpm, 4 C. SW28 rotors, using an Beckman XL-70 ultracentrifuge) 48 hours post infection and resuspended in DMEM w/o FCS (Invitrogen). Both concentrated supernatants as well as the same amount of just DMEM w/o FCS were incubated for 20 hours in the presence or absence of purified CD59his (Final concentration up to 100 ng/l, see example 1 and 2 for production and purification of CD59his)) at 37 C. under constant shaking. Viral particles were separated from not associated CD59his by ultracentrifugation as described above. The samples were then resuspended in DMEM w/o FCS post ultracentrifugation and aliquots used for immunoblotting (to assess association of CD59 to viral particles) or infecting confluent layers of CrFK cells kept in DMEM w/o FCS (to assess presence of viral particles post-painting by determining the cytopathic effectCPE).

Example 7: Production of mGFP-GPI

(20) To achieve expression of mGFP-GPI the sequence coding for the monomeric GFP as described by Zacharias et al (Zacharias (2002) was cloned into a vector backbone (pcDNA3.1hyg+ (Invitrogen)) providing the his-tag and the GSS of human decay accelerating factor (DAF, CD55) in a 2 step mutational PCR protocol, similar to the one explained in example 1. To primer sets were used: MEHindIIIF (5-cgcgcgcaagcttaatcaaaacatggctcagcggatgaca-3) SEQ ID No: 1 and MonoHisEG3R (5-gtggtggtgatggtggtgcttgtacagctcgtccatgccgagagt-3) SEQ ID No: 2 in the first set; HisEG1F (5-caccaccatcaccaccacccaaataaaggaagtggaacc-3) SEQ ID No: 3 and EGApaIR (5-gaatagggccctaagtcagcaagcccatg-3) SEQ ID No: 4 in a second set. Primers MEHindIIIF and EGApaIR were then used to amplify the complete sequence. The fragment was cloned into pcDNA3.1hyg+ (Invitrogen) using the unique HindIII and Apal sites. Transfection ofHEK293 cells was carried out as described in example 1. Purification and concentration of mG FP-GPI were carried out as described in example 2.

Example 8: Painting with Green Fluorescent Protein (GFP) Variant Proteins

(21) Viral particles were harvested from STAR cells (Ikeda et al. (2003)) as described previously (see example 3). Proteins were purified and concentrated as described previously (see example 2). Cell culture supernatants were concentrated as described previously (see example 3). Purified proteins were incubated with supernatant derived from 4 T 175 flasks per sample at final concentrations up to 100 ng/l protein. Painting reaction was allowed to commence for 20 hours at 37 C. under constant shaking before ultracentrifugation (as described previously, example 3). No magnetic pre-purification was necessary, as no endogenous GFP can contaminate the samples.

Example 9: Detection of CD59 and mGFP-GPI

(22) Samples were separated on precast 4-12% gradient gels (Invitrogen) under non-denaturing conditions in MES buffer at 100 V. Electroblotting onto PVDF membranes (GE Healthcare) was carried out at 1.1 mA/cm2 for 1 hour. Membranes were blocked overnight in 4% milk powder and 1% bovine serum albumin (Sigma-Aldrich) dissolved in TTBS (5% v/v Tween 20, 150 mM NaCl, 20 mM TrisHCl pH 8.0). Primary antibodies for CD59 (Serotec), p24 (Polymun) and EGFP (Invitrogen) were used at dilutions of 1:2000 and 1:1000 (EGFP), respectively. Secondary antibodies conjugated to horse radish peroxidase (DakoCytomation) against mouse and rabbit IgG were used at dilutions between 1:5000 and 1:10 000. Signal detection was carried out using the ECLplus kit (GE HealthCare)

Example 10: Silver Staining of Proteins

(23) Silver staining of protein extracts was carried out as previously described (Shevchenko et al. (1996). In brief: After fixing and washing, the polyacrylamide gels were sensitized in a 0.02% sodium thiosulfate solution for 1 minute. An aqueous 0.1% silver solution was used for the incubation before development in a sodium carbonate/formaldehyde solution. Color development was stopped by washing in 5% acetic acid in water.

Example 11: Magnetic Nanoparticles (MNP) Associate Specifically with Recombinant GPI Proteins and Allow Magnetic Manipulation

(24) GPI-anchored 6 histidine tagged green fluorescent protein or GPI anchored 6 histidine tagged CD59 was expressed in inHEK293 as described previously (see examples 1 and 7). In brief: after two-step mutagenesis PCR to introduce the 6 His tag resulting plasmids were transfected into HEK293 cells by lipofection (Invitrogen). Total cell extracts from expressing cells were mixed with iron based, phospholipid micelle nickel-nitrilo-acetate coated MNPs (Lim (2006); size of 5-10 nm or 50 nm diameter). For binding to target proteins and isolation, MNPs are added to total protein lysates after sonication and mixed for 4 hours at room temperature, then placed into a magnetic stand (Qiagen) and supernatant collected for further testing. Particles plus protein pellet is washed with wash buffer containing ImM Imidazole in I extraction Buffer (0.15M NaCl, 0.05 M Tris pH 7.5, 1% v/v NP40 (Sigma), 0.5% w/v Sodiumdeoxycholate (Sigma) and mixed by pipeting. This process is repeated twice so that three washing steps are performed in total. Bound protein-MNP can be then used for painting experiments or eluted using high concentrations of imidazole (500 mM) (and hence purified for further analysis). Cells were analysed by immunoblots using GFP specific antibodies (Invitrogen) and Coomassie staining of polyacrylamide gels. Levels of cellular protein are dramatically reduced by the purification step (as can be seen in the Coomassie staining, FIG. 7, bottom panel) and a large portion of the total amount target protein is recovered after purification (FIG. 7 lane A), when compared with the complete extract (FIG. 7, lane B)

(25) TABLE-US-00003 Primerused MEHindIIIF (5-cgcgcgcaagcttaatcaaaaca tggctcagcggatgaca-3) SEQIDNo:1 MonoHisEG3R (5-gtggtggtgatggtggtgcttgt acagctcgtccatgccgagagt-S) SEQIDNo:2 HisEGIF (5-caccaccatcaccaccacccaaa taaaggaagtggaacc-3) SEQIDNo:3 EGApaIR (5-gaatagggccctaagtcagcaag cccatg-3)SEQIDNo:4 CD59(2)FKHindIII (5-cacgacaagcttaccatgggaat ccaaggagggtctgtcctgtt-3) SEQIDNo:5 CD59(2)RApal (5-atgacgggcccttagggatgaag gctccaggctgctgccagaa-3) SEQIDNo:6 CD59FHis (5-catcaccatcaccatcacctgca gtgctacaactgtccta-3) SEQIDNo:7 CD59RHis (5-gtgatggtgatggtgatggctat gacctgaatggcagaag-3) SEQIDNo:8

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