CP149 HBV Plasmid Construction
A pET11c plasmid containing the Cp149 sequence was gratefully received from Prof. Adam Zlotnick of the Oklahoma University Health Sciences Center. In order to provide tight inducible control overexpression (pET11c was found to be constitutively expressed to an unacceptable degree), the HBV gene was amplified using the following primers: Cp149F 5′-AAG AAG GAG GAT ATA GGT CTC AC ATG GAC ATT GAC CCT-3′ and Cp149R 5′-TCG GGC TTT GTT AGC AGC CGG AAG CTT ATA CTA AAC AAC CGT-3′ The PCR product was sequentially digested with Hind III and Bsa I, and then ligated into pQE-60 plasmid (Qiagen) which had been sequentially digested with Hind III and Nco I. The resulting plasmid was transformed into competent M15MA cells harboring the pREP4 plasmid yielding the expression cells M15MA(pQE-60/HBV).
Site-Directed Mutagenesis of Cp149 HBV
Mutations were introduced into the coat protein of HBV by standard overlap PCR to remove the methionine at position 66 (M66S). The following general forward and reverse primers were used: Forward 5′-AGA ATT CAT TAA AGA GGA GAA ATT AAC-3′, Reverse 5′-CCA AGC TCA GCT AAT GCT TAT CCT-3′, M66SF 5′-GGA GAC TTA TCT ACT CTA GCT-3′, M66SR 5′-AGC TAG AGT AGA TAA GTC TCC-3′. The PCR products were sequentially digested with Hind III and NcoI and ligated into pQE-60 that had previously been digested with Hind III and Nco I. The resulting plasmids were transformed into competent M15MA cells yielding the following expression construct: M15MA(pQE-60/M66S HBV).
Insertion of the K16 M Qβ Coat Protein Gene into pQE-60
The coat protein gene for Qβ containing the point mutation K16 M was originally created in the pET-28b plasmid, which is not compatible with expression in M15 based Met auxotrophic E. coli cells. Therefore, the Qβ coat protein gene was amplified from the pET-28b plasmid using the following primers: QBF 5′-AAA GAG GAG AAA TTA AGG TCT CAC ATG GCA AAA TTA GAG ACT-3′ and QBR 5′-CCA AGC TCA GCT AAT TAA GCT TTA TTA ATA-3′ The PCR product was sequentially digested with Hind III and Bsa I. The digested PCR product was ligated into pQE-60 plasmid (Qiagen), which had been sequentially digested with Hind III and Nco I. The resulting plasmid was transformed into competent M15MA cells harboring the pREP4 plasmid yielding the expression cells M15MA(pQE-60/Qβ K16M).
Site-Directed Mutagenesis of K16 M Qβ
The following primers were used to introduce Met at position 93 (T93M), using standard overlap PCR as above: Forward 5′-AGA ATT CAT TAA AGA GGA GAA ATT AAC-3′, Reverse 5′-CCA AGC TCA GCT AAT GCT TAT CCT-3′, WTF 5′-GGA GAC TTA TCT ACT CTA GCT-3′, WTR 5′-AGC TAG AGT AGA TAA GTC TCC-3′. The PCR products were sequentially digested with Hind III and NcoI and ligated into pQE-60 that had previously been digested with Hind III and Nco I. The resulting plasmids were transformed into competent M15MA cells yielding the following expression construct: M15MA(pQE-60/WT Qβ) and M15MA(pQE-60/T93 M Qβ).
Synthesis of Unnatural Amino Acids (1 and 2)
Several short routes to azidohomoalanine (1
) have been reported in the literature (15
). We chose a simplified version of the method of Rappoport and co-workers (68
), since the starting lactone is commercially available in racemic form at modest cost and an enantiospecific synthesis is not required because E. coli
incorporates only the L
-isomer during protein expression. α-Amino-γ
-butyrolactone hydrobromide (5.02 g, 27.6 mmol) was refluxed in 1:1 HBr/glacial HOAc (250 mL) overnight (17−24 h). The HBr and acetic acid were then removed by rotary evaporation, yielding crude bromohomoalanine. The crude material was then dissolved in a solution of NaN3
(9.00 g, 138 mmol) in water (125 mL) and refluxed again overnight. The solution was evaporated to dryness and the residual tan solid resuspended in a minimal volume of 0.1 M HCl. The acidic solution was passed through a column of Dowex 50WX4−100 ion-exchange resin, washing with water and eluting the product with 1 M NH4
OH. The collected fractions were evaporated to dryness, yielding an oil. Compound 1
was obtained as a tan solid (1.49 g, 10.3 mmol, 37%) upon resuspending in a minimal volume of water and lyophilizing. The purity of the solid was determined by NMR using pyridine as an internal standard and generally was >85% (overall yields 32−38%). 1
H NMR (D2
O, 200 MHz, d1
= 10 s) δ
3.44 (t, 2H, CHCH2
), 3.07 (dd, 1H, CH
), 1.58 (m, 2H, CHCH2
). Homopropargylglycine (2
) was prepared as previously described (10
Incorporation of 1 into HBV and Qβ
A single of colony of cells expressing the desired HBV or Qβ construct was used to inoculate 5 mL of SOB media containing carbenicillin (100 μg/mL) and kanamycin (50 μg/mL) and was grown overnight. The resulting culture was transferred into 500 mL of M9 minimal media supplemented with all 20 amino acids, carbenicillin (100 μg/mL), and kanamycin (100 μg/mL) and allowed to grow for 8 h. Aliquots (25 mL) of the resulting culture were transferred into each of 11 flasks containing 500 mL of fresh minimal media supplemented with all 20 amino acids, carbenicillin (100 μg/mL) and kanamycin (100 μg/mL) and allowed to grow overnight. In the morning the cells were pelleted and resuspended in 500 mL of fresh M9 minimal media supplemented with all of the natural amino acids minus methionine. The cells were agitated at 37 °C for 30−40 min, and then transferred into new flasks of M9 minimal media supplemented with all of the natural amino acids minus methionine, IPTG (1 mM), carbenicillin (100 μg/mL), kanamycin (100 μg/mL), and azidohomoalanine (80 mg of racemate). After 6 h at 37 °C, the cells were harvested and stored at −80 °C.
For each preparation, 30−60 g of cells were allowed to thaw at room temperature and were resuspended in 50 mL distilled water. Cold lysis buffer (200 mL, 50 mM HEPES pH 8.0, 500 mM NaCl, 0.1 mg/mL DNase1, and 0.1 mg/mL RNaseA) was added and the cells were subjected to three cycles of sonication (2 min sonication and 2 min rest per cycle). Lysozyme (1 mg per mL of lysis buffer) was added and the solution was stirred in the cold room for 1 h. Insoluble cell debris was removed by centrifugation at 10 000 rpm for 30 min in a JA-10 rotor (Beckman).
For HBV, ammonium sulfate was added to the supernatant to a final concentration of 40% of saturation, and allowed to stir at room temperature for 30 min. The precipitated HBV coat protein was separated by centrifugation (10 000 rpm, 30 min, JA-10 rotor), and then was resuspended in 0.1 M potassium phosphate buffer (50 mL, pH 7.0). Any remaining insoluble material was removed by centrifugation (10 000 rpm, 15 min, JA-17 rotor). Sodium chloride was added to a final concentration of 0.5 M and the HBV coat proteins were allowed to assemble overnight. The resulting assembled VLPs were separated from smaller proteins by ultrapelleting (42 000 rpm, 6 h, 4 °C, 50.2Ti rotor, L90K ultracentrifuge). The pelleted material was resus-pended in 3−10 mL of 0.1 M potassium phosphate buffer (pH 7.0). Further purification was accomplished by the use of two successive sucrose gradient sedimentations (10−40% sucrose gradients in SW28 rotor at 28 000 rpm for 6 h at 4 °C.
For Qβ, PEG 8000 was added to the supernatant to a final concentration of 10% and the mixture was allowed to stir at room temperature for 30 min. The precipitated Qβ capsids were separated by centrifugation (10 000 rpm, 30 min, JA-10 rotor), and then resuspended in 0.1 M potassium phosphate buffer (50 mL, pH 7.0). Insoluble material was removed by centrifugation (10 000 rpm, 15 min, JA-17 rotor), and VLPs were isolated and purified as for HBV above, with final concentration by ultrapelleting.
Characterization of HBV and Qβ Particles
All protein preparations were analyzed by denaturing gel electrophoresis (4−12% NuPAGE Bis-Tris gel, Invitrogen) to estimate their purity; in all cases, the anticipated coat protein band constituted >95% of the intensity visualized by densitometry after Coomassie blue staining. HBV concentration was determined by absorbance at 280 nm, 1 mg/mL providing an absorbance value of 1.74. The concentrations of modified HBV and all Qβ particles were determined with the Modified Bradford Assay (Pierce, Inc.) and a BSA standard curve. Since HBV VLPs do not contain significant quantities of RNA, the ratio of absorbances at 260 nm vs 280 nm was 0.6−0.7. Qβ VLPs package random cellular nucleic acid, giving rise to A260/A280 values of 1.8−1.9. The presence of individual, aggregated, and disassembled particles was determined by size-exclusion chromatography on a Superose 6 column using an Akta Explorer (GE Healthcare) fast protein liquid chromatography (FPLC) instrument. Transmission electron microscopy was performed by applying particles at a concentration of 0.2 mg/mL to a carbon-coated Formvar transmission electron grid. The grids were stained with 2% uranyl acetate, and visualized in a Phillips CM120 transmission electron microscope.
VLP samples were processed for determination of derivatization site(s) by MALDI mass spectrometry as follows. Samples (100 μL, 1−2 mg/mL protein) were mixed with 300 μL of 8 M urea and 30 μL of 1 M DTT, and incubated at 37 °C for 1 h to allow the protein to denature. Iodoacetamide (1 M, 50 μL) was added to cap any free cysteines and the sample was again incubated at 37 °C for 1 h. DTT (30 μL) was added to quench unreacted capping reagent, and the samples were diluted to a final volume of 1.9 mL using 25 mM ammonium bicarbonate (pH 8.0). Each sample was digested with 30 μg of either trypsin or Glu-C protease overnight at 37 °C. The samples were then concentrated to approximately 300 μL, and urea was removed with Zip-tips (Microcon) prior to MALDI analysis.
Cryoelectron Microscopy and Analysis
Samples were prepared for CryoEM analysis by preservation in vitreous ice via rapid-freeze plunging onto plasma cleaned Cflat carbon film grids using a Vitrobot (FEI Co). Data collection was performed on Tecnai F20 electron microscopes (FEI Co.) operating at 120 keV using a dose of ~20e−
and a nominal underfocus of 0.8 to 3 μ
m utilizing the Leginon data collection software (69
). For the reconstructions, 735 micrographs of azidohomoalanine-incorporated HBV were collected at a nominal magnification of 80 000× at a pixel size of 0.14 nm at the specimen. 125 micrographs of azidohomoalanine-incorporated Qβ
particles were collected at a nominal magnification of 50 000× at a pixel size of 0.23 nm at the specimen. All micrographs were collected on a 4000 × 4000 CCD camera (Gatan Inc.). CryoEM analysis of all other Qβ
variants, including the transferrin-alkyne conjugated particles, was performed under the same conditions as the azidohomoalanine-incorporated HBV particles.
The contrast transfer function (CTF) for each micrograph was estimated using the Automated CTF Estimation (ACE) package (70
). 8906 HBV particles and 11 882 Qβ
particles were extracted from the collected data at a box size of 304 × 304 pixels and 180 × 180 pixels, respectively. The HBV particles were binned by a factor of 2 for the reconstruction. Phase correction of the single particles and subsequent three-dimensional refinement was carried out with the EMAN software package (71
). The amplitudes of the resulting refined structures were adjusted with the SPIDER software package (72
). Resolution of the final HBV and Qβ
densities were determined to be around 8 Å and 10 Å (respectively) according to 0.5 FSC criteria. Rigid-body docking of crystal structures into the reconstruction density and graphical representations were produced by the Chimera visualization software package (73
Copper Catalyzed Azide-Alkyne Cycloaddition Reactions
Purified azide-containing HBV or Qβ particles were pelleted out of solution (42 000 rpm, 6 h), the supernatant was removed, and residual buffer was allowed to drain away from the pellet. The viral pellet was then taken into a nitrogen-filled glovebox (without exposure to vacuum) and resuspended in a minimal volume of degassed 0.1 M Tris at pH 8.0. A small aliquot of the virus solution was removed from the glovebox and used to determine virus concentration. A typical conjugation reaction employed a VLP concentration of 2 mg/mL in 0.1 M Tris (pH 8.0) in a round-bottomed 2 mL Eppendorf tube (rather than conical tubes that do not provide good mixing upon gentle agitation). Fluorescein alkyne 6 (final concentration 1 mM) was added, followed by a freshly prepared buffer solution of Cu(I) triflate and 2 equiv of ligand 4 or 5. Final concentrations of Cu were either 100 μM or 250 μM for HBV, 500 μM or 1 mM for Qβ. The tube was sealed, placed in a secondary sealed container (usually a small round-bottomed flask), removed from the glovebox, and attached to a slow tumbler arm for agitation at room temperature for 16−18 h. After completion of the reaction, intact virus-like particles were separated from excess labeling reagent using a combination of 10−40% sucrose gradients (38 000 rpm, SW41 rotor, 4 h for HBV, 3 h for Qβ) and ultrapelleting.