Sypro Ruby

For purification of dynein complexes, a frozen pellet of 250-ml insect cell culture was thawed on ice and resuspended in lysis buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 1 mM DTT, 0.1 mM ATP, 10% (v/v) glycerol, 2 mM PMSF) supplemented with protease inhibitors (Complete-EDTA Free, Roche Applied Science) to a final volume of 25 ml. Cells were lysed in a 40-ml dounce-type tissue grinder (Wheaton) using 20–30 strokes. The lysate was cleared by centrifugation (504,000 g, 45 min, 4°C; Type 70 Ti Rotor, Beckman Coulter) and added to 3–5 ml pre-washed IgG Sepharose 6 FastFlow beads (GE Healthcare) in a 2.5 × 10 cm Econo-Column (Bio-Rad) and incubated on a roller for 2–6 h. After incubation, the dynein complexes bound to IgG Sepharose beads were washed with 50 ml lysis buffer and 50 ml TEV buffer (50 mM Tris–HCl pH 7.4, 148 mM KAc, 2 mM MgAc, 1 mM EGTA, 10% (v/v) glycerol, 0.1 mM ATP, 1 mM DTT). To fluorescently label the SNAPf tag, dynein coated beads were incubated with either SNAP-Cell TMR-Star or SNAP-Surface Alexa Fluor 647 substrate (New England Biolabs) as described below (see also Supplementary Fig S5). Subsequently, the beads were resuspended in TEV buffer (final volume 5–15 ml) with 50–100 μl TEV protease (4 mg/ml) and incubated at 4°C on a roller overnight. After TEV cleavage, the beads were removed and the protein of interest concentrated in a 100 K molecular weight cut-off concentrator (Amicon Ultracel, Merck-Millipore) to 1–5 mg/ml. TEV protease was removed by size-exclusion chromatography using a TSKgel G4000SW_XL column with a TSKgel SW_XL guard column (TOSOH Bioscience) equilibrated in GF150 buffer (25 mM HEPES pH 7.4, 150 mM KCl, 1 mM MgCl₂, 5 mM DTT, 0.1 mM ATP) or a Superose 6 PC 3.2/30 equilibrated in GF50 buffer (25 mM HEPES pH 7.4, 50 mM KCl, 1 mM MgCl₂, 5 mM DTT, 0.1 mM ATP) using an Ettan LC system (GE Healthcare). Peak fractions were collected, pooled and concentrated to 0.5–10 mg/ml using Amicon concentrators as described above. All purification steps were performed at 4°C. The purification of native pig brain dynein, dynactin and recombinant BICD2N is described in the Supplementary Information.
SDS–PAGE was performed using Novex 4–12% Bis–Tris precast gels using either MOPS or MES buffer (Life Technologies). Gels were stained with either the Coomassie-based reagent Instant Blue (Expedeon) or SYPRO Ruby (Life Technologies) and imaged using a Gel Doc XR+ system with Image Lab 4.0 software (Bio-Rad). Protein concentrations were measured using Quick Start Bradford dye (Bio-Rad) and an Ultrospec 2100 Pro spectrophotometer (Amersham). The proteins were flash frozen in liquid nitrogen and stored at −80°C. Dynein was frozen in the presence of approximately 10% (v/v) glycerol.

MCV reporter vector stocks were produced by transfecting human embryonic kidney cells engineered to stably express the cDNA of SV40 T antigen (293TT) [15] (link). The cells were transfected using Lipofectamine2000 (Invitrogen) according to previously-reported methods [42] . In initial studies, plasmids pwM and ph2m [12] expressing, respectively, codon-modified versions of the VP1 and VP2 genes of MCV strain 339, were co-transfected with a GFP reporter plasmid, pEGFP-N1 (Clontech). Neutralization assay stocks employed phGluc, which encodes a Gaussia luciferase reporter gene (NEB), as a reporter plasmid. Forty-eight hours after transfection, the cells were harvested and lysed at high density (10⁸ cells per ml) in Dulbecco's phosphate buffered saline (DPBS, Invitrogen) supplemented with 9.5 mM MgCl₂, 0.4% Triton X-100 (Pierce), 0.1% RNase A/T1 cocktail (Ambion) and antibiotic-antimycotic (Invitrogen). The cell lysate was incubated at 37°C overnight with the goal of promoting capsid maturation [43] (link). Lysates containing mature capsids were clarified by centrifugation for 10 min at 5000×g. The clarified supernatant was loaded onto a 27–33–39% iodixanol (Optiprep, Sigma) step gradient prepared in DPBS with a total of 0.8 M NaCl. The gradients were ultracentrifuged 3.5 hours in an SW55 rotor at 50,000 rpm (234,000×g). Gradient fractions were screened for the presence of encapsidated DNA using Quant-iT Picogreen dsDNA Reagent (Invitrogen). VP1 protein concentration was determined by comparing vector stock to bovine serum albumin standard (BioRad) in SYPRO Ruby-stained Nupage gels (Invitrogen). Vector stock yields were typically several µg of purified VP1 per 225 cm² flask of transfected cells.
Vector stocks based on murine polyomavirus (MPyV) or BKV were produced using a similar scheme. For MPyV cells were co-transfected with plasmids pwP and ph2p [12] (carrying codon-modified MPyV VP1 and VP2, respectively) together with phGluc. An additional plasmid, ph3p, encoding the MPyV minor capsid protein VP3, was also included in the co-transfection mixture. For BKV vector stocks, plasmid pCAG-BKV (a generous gift from Dr. Akira Nakanishi (NCGG, Japan) [28] (link)) encoding the capsid protein genes was co-transfected with phGluc.
In some virion production systems, capsids containing linear fragments of cellular DNA can substantially outnumber capsids containing the viral genome or desired reporter plasmid [43] (link),[44] (link). In the vector harvest procedure detailed above, unwanted capsids associated with large segments of cellular DNA (as opposed to reporter plasmid DNA) tend to sediment away during the 5000×g clarification step and tend to be retained toward the top of the Optiprep gradient ([42] and unpublished results). For production of VLPs, recovery of capsids containing cellular DNA is desirable and was achieved by adding Benzonase (Sigma) and Plasmid Safe (Epicentre) nucleases to the lysis buffer (0.1% each) and adjusting the lysate to 0.8 M NaCl immediately prior to clarification. These modifications to the harvest protocol increased VLP yield to roughly 1 mg of VP1 per transfected 225 cm² flask.
Maps of plasmids used in this work and detailed virus production protocols are available from our laboratory website <http://home.ccr.cancer.gov/LCO/>.

Synthetic gene variants and chimeras were all made by standard in-house procedures essentially as previously described [45] (link) and cloned into a pET24a expression vector (EMD, Madison, WI) under control of the T7 promoter, between the XbaI and EcoRI restriction sites. Each construct was completely sequenced in both directions to ensure consistency with the designed sequence. Each variant plasmid was transformed into E. coli expression host strain BL21(DE3) pLysS (Invitrogen, Carlsbad, CA). BL21(DE3) pLysS was chosen as the host for all expression studies described. The low-level expression in this host of T7 lysozyme, an inhibitor of T7 RNA polymerase, gives tight repression of heterologous expression prior to induction to minimize potential gene toxicity which could affect data quality.
Prior to protein expression analysis of the variants, expression was analyzed for multiple variants showing a range of expression levels to determine appropriate expression time and temperature. Strong, consistent expression was achieved at 30°C, a commonly used temperature for heterologous expression in E. coli. Time courses at 30°C showed expressed protein levels increasing to a maximum after approximately two hours, as the cells entered stationary phase growth, and expression remained steady for at least five hours. Relative protein expression levels between these variants were consistent as protein accumulated during growth phase and were maintained in stationary phase (data not shown). For our variant analysis we chose to express for four hours at 30°C.
At least three independent isolates for each gene were picked and cultured overnight in 2 ml Luria Broth (LB) containing 25 µg/ml kanamycin and 25 µg/ml chloramphenicol. Overnight cultures were diluted 50-fold in fresh media and incubated at 37°C until the cells were in mid-log growth (OD at 600 nm = 0.6). Expression was induced by addition of IPTG to 1 mM and incubation for four hours at 30°C. Final optical densities of cultures were measured and equivalent amounts of culture were analyzed by polyacrylamide gel electrophoresis. Gels were stained with Sypro Ruby (Pierce), visualized by fluorescence imaging, and protein band intensities quantified using TotalLab100 image analysis software (Nonlinear, Inc). Each gel contained protein concentration standards to calibrate band intensity. In each experiment, a consistent reference variant was co-expressed in triplicate. For analysis of polymerase expression, the reference was a phi29 DNA polymerase variant identical to variant 21 but containing two differences in the 5′ untranslated region. For analysis of the scFv variants, Variant A13 was used as the reference. Reference variants were used to correct for experiment to experiment variation in yield. Measured expression levels are all relative to these references. Reported values in µg/ml are normalized to the average expression level of the references over the sum of experiments. The detection limit of the assay was approximately 5 µg protein per ml culture at an A₆₀₀ = 3. The standard error of measured expression for variant repeats was generally <20% of the mean.

Clarified rRBD supernatant samples from the transient and stable fed-batch cultures were purified for gel analysis using a Ni-NTA Spin Kit (Qiagen) under native conditions. Spin columns were equilibrated, loaded with 3 × 600 µL harvested supernatant, washed, and eluted per the manufacturer’s protocol. Samples were buffer exchanged into PBS using 10 kDa Amicon Ultra-0.5 Centrifugal Filter Units (MilliporeSigma). rRBD concentrations were measured by A280 absorbance.
Purified rRBD samples were buffer exchanged into molecular biology grade water (Fisher Scientific) using 10 kDa Amicon Ultra-0.5 Centrifugal Filter Units (MilliporeSigma). rRBD concentrations were measured by A280 absorbance. 15 µg of rRBD was then denatured and digested with PNGaseF (Promega) based on the manufacturer’s protocol. In brief, rRBD was denatured at 95 °C for 10 min in the presence of dithiothreitol (DTT; New England Biolabs) and sodium dodecyl-sulfate (SDS; Bio-Rad). PNGase F digestion with 1U/µL enzyme was performed in the presence of sodium phosphate pH 7.4 (Fisher Scientific) and Triton X-100 (MilliporeSigma) at 37 °C for 4 h.
Purified rRBD samples diluted with 2x Laemmli sample buffer (Bio-Rad) supplemented with 120 mM DTT (New England Biolabs) were boiled for 5–10 min. 1 µg of intact protein or PNGaseF digested protein was loaded into a 4–15% Mini-PROTEAN® TGX™ gel (Bio-Rad). 5 µL of Precision Plus Protein™ Unstained Protein Standards (Bio-Rad) was used as a molecular weight marker. The gel was run for 50 min at 150 V and then stained with SYPRO® Ruby (ThermoFisher). Fix and wash buffers were prepared per the manufacturer’s protocol, and a modified rapid protocol was executed for gel fixing, staining, and washing while rocking the gel on an orbital shaker. In brief, the gel was shaken in fresh fixing buffer 2 × 15 min, stained for 3 h, and shaken in fresh wash buffer 2 × 15 min. Gels were imaged on a Typhoon FLA 7000 (Cytiva) using the 473 nm laser and 580 nm emission filter.

Whole-cell lysate samples were immediately frozen after treatment completion. Membrane/cytosol fractionated lysates were processed immediately after the treatment without a freezing step. Further details of homogenization and western blotting technique can be consulted in Cilleros-Mañé et al. [41 (link)].
The densitometry of the bands was obtained with ImageJ software. The integrated optical density of the bands was normalized with respect to: (1) the background values; and to (2) the total protein transferred on PVDF membranes, measured by total protein analysis (Sypro Ruby protein blot stain, Bio-Rad [49 (link)]). The relative variations between the experimental samples and the control samples were calculated from the same membrane image. All presented data derive from densitometry measurements made of three to ten separate replicates, plotted against controls. Data quantification was performed blindly.

Kinase assays were performed with HA₂-Pho85- and HA₂- Pho85^E53A -bound beads, as described in [31 (link)]. The reaction was performed in kinase buffer (50 mM Tris-HCl pH 7.5, 20 mM MgCl₂, 1 mM DTT). The reaction was carried out with 50 ng of kinase and Pho80 and 40 ng of the substrate. By adding the ATP mix (final concentration in reaction: 1mM ATP, 10 μCi γ-[32^P]-ATP) the reaction was started and performed for 30 min at 30°C. By adding 2X SDS-PAGE sample buffer, the reaction was stopped. Samples were denatured at 65°C for 10 min, proteins were separated by SDS-PAGE, stained with Sypro Ruby (Invitrogen, Thermo Fisher Scientific, Basel, Switzerland) to assess loading, and analyzed using a phospho-imager (Typhoon FLA 9500; GE Healthcare, Opfikon, Switzerland), as described in [22 (link)].