The overall goal of the following experiment is to perform a solvent accessibility analysis of cystine residues on the surface of virus-like particles or vlp, followed by cross-linking of peptides to cystines on the virus-like particles. This is achieved by first cloning the maze Reto pheno virus or MRM RF VCO protein in a potato virus X based virus vector. Next and benanna plants are inoculated with the modified virus and MRM RFVV PS formed from the coat protein are purified.
Finally, VLP and peptide are cross-linked using ammonia PEG four ide and detected by western blot. Results are obtained that identify the production of VLP peptide complexes through western blot analysis. The technique extends the knowledge base on chemically modified surface of viruses and virus-like particle to produce new material for nanotechnology.
After producing Capped T seven RNA transcripts from potato virus X base vector plasmids carrying mrm, RFV wild type and CIS mutated coat protein genes use two aliquots of 10 microliters each to inoculate two and benam leaves incubate the plants for 10 days in a greenhouse at 60%humidity and 16 hours of light at 25 degrees Celsius and eight hours of dark at 20 degrees Celsius. At the end of the incubation harvest the virus infected leaves, weigh the plant tissue and place it on ice. After purifying the virus-like particles according to the text protocol, prepare a one millimolar solution.
A fluorescein five IDE in 50 millimolar sodium phosphate buffer, pH 7.01 millimolar EDTA vortex to completely dissolve and use aluminum foil to protect the solution from light. Mix approximately 30 microliters of one microgram per microliter of VLP and 60 microliters of the fluorescein five IDE and incubate for two hours at room temperature or overnight at four degrees Celsius. Terminate the reaction by adding DTT out of final concentration of 50 millimolar using a desalting spin column.
Remove the non-reactive fluorescein to visualize the vlp with available cysteine residues for cross-linking. Perform SDS page analysis by adding 10 microliters of two x sample buffer to 10 microliters of each reaction. Store the labeled protein protected from light at four degrees Celsius for up to one month or in single use aliquots at negative 20 degrees Celsius up to three months.
Prepare a Fresh crosslinking stock solution by dissolving 10 milligrams of NHS Peg four IDE crosslinker in 680 microliters of dimethyl sulfoxide dissolve a 17 F amino acid lung peptide in 50 microliters of conjugation buffer at a concentration of 0.1 millimolar. Add four microliters of crosslinker to 50 microliters of the dissolved peptide for a one millimolar final concentration. Incubate the reaction for two hours at four degrees Celsius.
Then use a desalting column to purify the reaction using vlp with available cysteine residues for cross-linking and the cross-linked peptide. Set up a reaction mix at a molar ratio determined by the number of files and activated amines involved in the reaction and the difference in molecular weight between vlp and peptide. After incubating the reaction for two hours at room temperature, use it to carry out SDS page and western blotting according to the text protocol.
An example Of reactive amino acids of the MRF VCO protein is shown here. Each amino acid selected can be used to attach moieties if it is surface exposed. The chemical techniques include traditional bio conjugation strategies such as the isolation of the amino group of lysine, alkylation of the sulf hydrolic group of cystine, and activation of carboxylic acid residues and coupling with added amines.
In addition, the aromatic groups of tyrosine and tryptophan can be attractive targets for bio conjugation through ization and alkylation illustrated. Here is the solvent accessibility of CIS residues of VLP to file specific reagents purified wild type vlp and CIS VLP mutants carrying cystine residues in position 107 to 111 and 192 to 194 of the CP gene are unreactive in native conditions with a THI specific reagent fluorescein five IDE imparts orange fluorescence only for the cyst VLP mutant carrying cystine residues in position 125 to 129 of the C gene. The result shows that VLP in lane two have a geometric arrangement of cystine residues resulting in solvent exposed free file groups, whereas the cystine residues in the samples in lane one and three are perhaps involved in diss sulfide bridges.
In the folding of the coat protein labeling reactions with IDE PEG two biotin followed by a biotin elation assay represent another example of analysis of solvent accessibility of cis residues. Because biotin is a relatively small molecule, it can be conjugated in several copies on the vlp, each of which combined one molecule of avadon as shown here. The level of biotin, 84.74 mole of biotin per mole of protein indicates the number of the biotin molecules attached to the vlp, and consequently the number of ins that can be displayed on the exterior surface because of the availability of several file groups on the vlp peptides can be attached by crosslinking reactions with NHS Peg four ide.
This reagent is a hetero bifunctional crosslinker, which contains reactive ends such as n hydroxy CIN aide or NHS Ester and IDE groups allowing covalent conjugation of amine and hyd containing molecules. In this example, the NHS Ester reacts with primary amines forming amide bonds, whereas IDE reacts with sulf hydro groups forming stable thioester bonds. As seen here, cross-linking reactions produce VLP peptide complexes that are immunoreactive with the specific antibodies in western blot.
This development demonstrates to researchers in the field of virology how to explore the use of virus particles as a scaffold for the attachment of a large variety of ligands in a regular geometric array for numerous applications.
