Editing VLP Expression and Purification (section)

== Cytos Patent Method ==

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E. coli JM109 was transformed with Qβ coat protein expressing plasmids. 5 ml of LB liquid medium containing 20 μg/ml ampicillin was inoculated with a clone transformed with Qβ coat protein expression plasmid. The inoculated culture was incubated at 37° C. for 16-24 hours without shaking. The culture was subsequently diluted 1:100 in 100-300 ml of fresh LB medium, containing 20 μg/ml ampicillin, and incubated at 37° C. overnight without shaking. The resulting second inoculum was diluted 1:50 in M9 medium containing 1% Casamino acids and 0.2% glucose in flasks, and incubated at 37° C. overnight under shaking. The cells were pelleted by centrifugation and frozen for storage.

Purification

Solutions and buffers for the purification procedure:

1. Lysis buffer LB

50 mM Tris-HCl pH8.0 with 5 mM EDTA, 0.1% tritonX100 and freshly prepared PMSF at a concentration of 5 micrograms per ml without lysozyme and DNAse. 2. Saturated ammonium sulphate in water (SAS)

3. Buffer NET.

20 mM Tris-HCl, pH 7.8 with 5 mM EDTA and 150 mM NaCl.

4. PEG

40% (w/v) polyethylenglycol 6000 in NET

Disruption and lysis

Frozen cells were resuspended in LB at 2 ml/g cells. The mixture was sonicated with 22 kH five times for 15 seconds, with intervals of 1 min to cool the solution on ice. The lysate was then centrifuged at 14000 rpm, for 1 h using a Janecki K 60 rotor. The centrifugation steps described below were all performed using the same rotor, except otherwise stated. The supernatant was stored at 4° C., while cell debris was washed twice with LB. After centrifugation, the supernatants of the lysate and wash fractions were pooled.

Fractionation

A saturated ammonium sulphate solution was added drop wise under stirring to the above pooled lysate. The volume of the SAS was adjusted to be one fifth of total volume, to obtain 20% of saturation. The solution was left standing overnight, and was centrifuged the next day at 14000 rpm, for 20 min. The pellet was washed with a small amount of 20% ammonium sulphate, and centrifuged again. The obtained supernatants were pooled, and SAS was added drop wise to obtain 40% of saturation. The solution was left standing overnight, and was centrifuged the next day at 14000 rpm, for 20 min. The obtained pellet was solubilised in NET buffer.

Chromatography

The capsid or VLP protein resolubilized in NET buffer was loaded on a Sepharose CL-4B column. Three peaks eluted during chromatography. The first one mainly contained membranes and membrane fragments, and was not collected. VLPs were contained in the second peak, while the third one contained other E. coli proteins.

The peak fractions were pooled, and the '''NaCl''' concentration was adjusted to a final concentration of '''0.65 M'''. A volume of PEG solution corresponding to one half of the pooled peak fraction was added drop wise under stirring. The solution was left to stand overnight without stirring. The capsid protein was sedimented by centrifugation at 14000 rpm for 20 min. It was then solubilized in a minimal volume of NET and loaded again on the Sepharose CL-4B column. The peak fractions were pooled, and precipitated with ammonium sulphate at 60% of saturation (w/v). After centrifugation and resolubilization in NET buffer, capsid protein was loaded on a Sepharose CL-6B column for rechromatography.

Dialysis and Drying

The peak fractions obtained above were pooled and extensively dialysed against sterile water, and lyophilized for storage.



Preparation of Qβ VLPs of the Invention by Disassembly/Reassembly in the Presence of Different Polyanionic Macromolecules Resulting in Reassembled Qβ VLPs (A) Disassembly of Prior Art Qβ VLP

45 mg prior art Qβ VLP (2.5 mg/ml, as determined by Bradford analysis) in PBS (20 mM Phosphate, 150 mM NaCl, pH 7.5) purified from E. coli lysate was reduced with '''10 mM DTT''' for 15 min at room temperature under stirring conditions. '''Magnesium chloride '''was then added to'''0.7 M''' final concentration and the incubation was continued for 15 min at room temperature under stirring conditions, which led to the precipitation of the encapsulated host cell RNA. The solution was centrifuged for 10 min at 4000 rpm at 4° C. (Eppendorf 5810 R, in fixed angle rotor A-4-62 used in all following steps) in order to remove the precipitated RNA from the solution. The supernatant, containing the released, dimeric Qβ coat protein, was used for the chromatographic purification steps.

(B) Purification of the Qβ Coat Protein by Cation Exchange Chromatography and by Size Exclusion Chromatography

The supernatant of the disassembly reaction, containing the '''dimeric '''coat protein, host cell proteins and residual host cell RNA, was diluted 1:15 in water to adjust conductivity below 10 mS/cm and was loaded onto a'''SP-Sepharose FF '''column (xk 16/20, 6 ml, Amersham Bioscience). The column was equilibrated beforehand with 20 mM sodium phosphate buffer pH 7. The elution of the bound coat protein was accomplished by a step gradient to 20 mM sodium phosphate/500 mM sodium chloride and the protein was collected in a fraction volume of approx. 25 ml. The chromatography was carried out at room temperature with a flow rate of 5 ml/min and the absorbance was monitored at 260 nm and 2800 nm.

In the second step, the isolated Qβ coat protein (the eluted fraction from the cation exchange column) was loaded (in two runs) onto a Sephacryl S-100 HR column (xk 26/60, 320 ml, Amersham Bioscience), equilibrated with 20 mM sodium phosphate/250 mM sodium chloride; pH 6.5. The chromatography was carried out at room temperature with a flow rate of 2.5 ml/min and the absorbance was monitored at 260 nm and 280 nm. Fractions of 5 ml were collected.

(C1) Reassembly of the Qβ VLP by Dialysis

Purified Qβ coat protein (2.2 mg/ml in 20 mM sodium phosphate pH 6.5), one polyanionic macromolecule (2 mg/ml in water), urea (7.2 M in water) and DTT (0.5 M in water) were mixed to the final concentrations of 1.4 mg/ml coat protein, 0.14 mg/ml of the respective polyanionic macromolecule, 1 M urea and 2.5 mM DTT. The mixtures (1 ml each) were dialyzed for 2 days at 5° C. in 20 mM Tris HCl, 150 mM NaCl pH 8, using membranes with 3.5 kDa cut off. The polyanionic macromolecules were: polygalacturonic acid (25000-50000, Fluka), dextran sulfate (MW 5000 and 10000, Sigma), '''poly-L-aspartic acid''' (MW 11000 and 33400, Sigma), p'''oly-L-glutamic acid''' (MW 3000, 13600 and 84600, Sigma) and'''tRNAs from bakers yeast and wheat germ'''.

(C2) Reassembly of the Qβ VLP by Diafiltration

33 ml purified Qβ coat protein (1.5 mg/ml in 20 mM sodium phosphate pH 6.5, 250 mM NaCl) was mixed with water and urea (7.2 M in water), NaCl (5 M in water) and poly-L-glutamic acid (2 mg/ml in water, MW: 84600). The volume of the mixture was 50 ml and the final concentrations of the components were 1 mg/ml coat protein, 300 mM NaCl, 1.0 M urea and 0.2 mg/ml poly-L-glutamic acid. The mixture was then diafiltrated at room temperature, against 500 ml of 20 mM Tris HCl pH 8, 50 mM NaCl, applying a cross flow rate of 10 ml/min and a permeate flow rate of 2.5 ml/min, in a tangential flow filtration apparatus using a Pellicon XL membrane cartridge (Biomax 5K, Millipore).

Analysis of the Reassembled Qβ-VLPs

(D1) Formation of Disulfide Bonds in the Qβ-VLPs

The reassembled Qβ-VLPs in the presence of different polyanionic macromolecules as described in EXAMPLE 4(C1) and 4(C2) were analyzed by non-reducing SDS-PAGE and compared to the prior art Qβ VLP. The reassembled Qβ-VLPs exhibited bands of disulfide-linked pentameric and hexameric forms of the coat protein; similar to the prior art Qβ VLP, indicating the correct structural arrangement of the coat protein units in the reassembled QβVLP (FIG. 2).

(D2) Hydrodynamic Size of the Qβ VLPs Reassembled in the Presence of Different Polyanionic Macromolecules by Analytical Size Exclusion Chromatography

Samples of the Qβ-VLPs reassembled in the presence of different polyanionic macromolecules as described in EXAMPLE 4(C1) and 4(C2) were analyzed by analytical size exclusion chromatography and compared to the prior art Qβ VLP. The reassembled Qβ VLP showed a peak migrating at the same retention time as the peak representing the prior art QβVLP, indicating the overall size and structure of the reassembled VLP is the same as the prior art VLP.

Example 5 In Vitro Assembly of AP205 VLPs (A) Purification of AP205 Coat Protein

Disassembly: 20 ml of AP205 VLP solution (1.6 mg/ml in PBS, purified from E. coli extract) was mixed with 0.2 ml of 0.5 M DTT and incubated for 30 min at room temperature. 5 ml of 5 M NaCl was added and the mixture was then incubated for 15 min at 60° C., causing precipitation of the DTT-reduced coat proteins. The turbid mixture was centrifuged (rotor Sorvall SS34, 10000 g, 10 min, 20° C.) and the supernatant was discarded and the pellet was dispersed in 20 ml of 1 M Urea/20 mM Na Citrate pH 3.2. After stirring for 30 min at room temperature, the dispersion was adjusted to pH 6.5 by addition of 1.5 M Na2HPO4 and then centrifuged (rotor Sorvall SS34, 10000 g, 10 min, 20° C.) to obtain supernatant containing dimeric coat protein.

Cation exchange chromatography: The supernatant (see above) was diluted with 20 ml water to adjust a conductivity of approx. 5 mS/cm. The resulting solution was loaded on a column of 6 ml SP Sepharose FF (Amersham Bioscience) which was previously equilibrated with 20 mM sodium phosphate pH 6.5 buffer. After loading, the column was washed with 48 ml of 20 mM sodium phosphate pH 6.5 buffer followed by elution of the bound coat protein by a linear gradient to 1 M NaCl over 20 column volumes. The fractions of the main peak were pooled and analyzed by SDS-PAGE and UV spectroscopy. According to SDS-PAGE, the isolated coat protein was essentially pure from other protein contaminations. According to the UV spectroscopy, the protein concentration was 0.6 mg/ml (total amount 12 mg), taking that 1 A280 unit reflects 1.01 mg/ml of AP205 coat protein. Furthermore, the value of A280 (0.5999) over the value of A260 (0.291) is 2, indicating that the preparation is essentially free of nucleic acids.

(B) Assembly of AP205 VLPs

Assembly in the absence of any polyanionic macromolecule: The eluted protein fraction from above was diafiltrated and concentrated by TFF to a protein concentration of 1 mg/ml in 20 mM sodium phosphate pH 6.5. 500 μl of that solution was mixed with 50 μl of 5 M NaCl solution and incubated for 48 h at room temperature. The formation of reassembled VLPs in the mixture was shown by non-reducing SDS-PAGE and by size exclusion HPLC (FIG. 5A). A TSKgel G5000 PWXL column (Tosoh Bioscience), equilibrated with 20 mM sodium phosphate, 150 mM NaCl pH 7.2, was used for the HPLC analysis.

Assembly in the presence of '''polyglutamic acid''': 375 μl of purified AP205 coat protein (1 mg/ml in 20 mM sodium phosphate pH 6.5) was mixed with 50 μl of NaCl stock solution (5 M in water) solution, 50 μl of polyglutamic acid stock solution (2 mg/ml in water, MW: 86400, Sigma) and 25 μl of water. The mixture was incubated for 48 h at room temperature. The formation of reassembled VLP in the mixture was shown by non-reducing SDS-PAGE and by size exclusion HPLC (FIG. 5B). The coat protein in the mixture was almost completely incorporated into the VLPs, showing a higher assembly efficiency than the AP205 coat protein assembled in the absence of any polyanionic macromolecule (FIG. 5A).

Example 6 Preparation of the Reassembled fr VLP or the Reassembled GA in the Presence of Polyanionic Macromolecules Similar experimental conditions as disclosed in Example 1 and 4 are applied to recombinantly produce prior art fr or GA VLP in E. coli, purify and disassemble these VLPs, and purify the obtained coat proteins of the corresponding RNA phages. Similar experimental conditions are then applied to reassemble the coat proteins into the reassembled fr VLP or the reassembled GA VLP.

Assembly of VLP from Purified Fusion Protein.

Qβ-Aβ1-6 fusion protein (2.2 mg/ml in 20 mM sodium phosphate pH 6.5), one polyanionic macromolecule (2 mg/ml in water), urea (7.2 M in water) and DTT (0.5 M in water) are mixed to the final concentrations of 1.4 mg/ml fusion protein, 0.14 mg/ml of the respective polyanionic macromolecule, 1 M urea and 2.5 mM DTT. The mixtures (1 ml each) are dialyzed for 2 days at 5° C. in 20 mM Tris HCl, 150 mM NaCl pH 8, using membranes with 3.5 kDa cut off. The polyanionic macromolecules are: polygalacturonic acid (25000-50000, Fluka), poly-L-glutamic acid (MW 3000, 13600 and 84600, Sigma) or tRNAs from bakers yeast or wheat germ.

 Purified fr AP 1-6 fusion protein (2.2 mg/ml in 20 mM sodium phosphate pH 6.5), one polyanionic macromolecule (2 mg/ml in water), urea (7.2 M in water) and DTT (0.5 M in water) are mixed to the final concentrations of 1.4 mg/mil fusion protein, 0.14 mg/ml of the respective polyanionic macromolecule, 1 M urea and 2.5 mM DTT. The mixtures (1 ml each) are dialyzed for 2 days at 5° C. in 20 mM Tris HCl, 150 mM NaCl pH 8, using membranes with 3.5 kDa cut off. The polyanionic macromolecules are: polygalacturonic acid (25000-50000, Fluka), dextran sulfate (MW 5000 and 10000, Sigma), poly-L-aspartic acid (MW 11000 and 33400, Sigma).

Peptide

For peptides Angio 1 to Angio 4: A solution of 5 ml of the reassembled 2 mg/ml AP205 or GA VLP in 20 mM Hepes. 150 mM NaCl pH 7.4 is reacted for 30 minutes with 507 μl of a solution of 13 mg/ml Sulfo-MBS (Pierce) in H2O at 25° C. on a rocking shaker. The reaction solution is subsequently dialyzed twice for 2 hours against 2 L of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. 665 μl of the dialyzed reaction mixture is then reacted with 2.8 μl of each of the corresponding 100 mM peptide stock solution (in DMSO) for two hours at 25° C. on a rocking shaker. The reaction mixture is subsequently dialyzed 2×2 hours against 2 liters of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C.

For peptides Angio 5-9 and Angio 13-15: A solution of 3 ml of the reassembled 2 mg/ml AP205 or GA VLP in 20 mM Hepes. 150 mM NaCl pH 7.2 is reacted for 50 minutes with 86 μl of a solution of 100 mM SMPH (succinimidyl-6-(β-maleimidopropionoamido hexanoate, Pierce) in DMSO at 25° C. on a rocking shaker. The reaction solution is subsequently dialyzed twice for 2 hours against 2 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. 514 μl of the dialyzed reaction mixture is then reacted with 3.6 μl of each of the corresponding 100 mM peptide stock solution (in DMSO) for 4 hours at 25° C. on a rocking shaker. The reaction mixture is subsequently dialyzed 2×2 hours against 2 liters of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C.