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Additional Supporting Information may be found in the online version of this article.

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BTPR_1655_sm_SuppFig1.tif506KSupporting Information Figure 1: RBP binds specifically to RCNMV and not the secondary detection reagent. Yeast cells expressing RBP as cell surface fusions were labeled with 10 nM biotinylated RCNMV (black histogram) or PBS-BSA only (grey histogram), followed by secondary labeling with strep-PE, and analyzed using flow cytometry. The higher PE fluorescence in the presence of RCNMV (black histogram) shows specific binding of yeast cell surface displayed RBP to RCNMV.
BTPR_1655_sm_SuppFig2.tif1645KSupporting Information Figure 2: RCNMV binding by RBP recovered after avidity-mediated separation and degradation of viral coat protein. 40 μg each of fresh RBP or RBP recovered from the supernatant in ultracentrifugation step (from experiment in Figure 5) were immobilized on Ni-NTA magnetic beads through overnight incubation at 4 °C. Beads were washed in PBS-BSA and labeled with 30 nM biotinylated RCNMV, followed by secondary labeling with strep-PE, and analyzed by flow cytometry. (a) Fluorescence histogram of control beads (without immobilized RBP) labeled with strep-PE only. (b) Fluorescence histogram of beads with fresh RBP immobilized, and labeled with biotinylated RCNMV followed by strep-PE. (c) Fluorescence histogram of beads coated with recycled RBP followed by labeling with RCNMV and strep-PE. PE fluorescence in the same order of magnitude seen in both (b) and (c) demonstrates that RBP recovered in the supernatant retains RCNMV-binding activity (d) Avidity-mediated separation of RCNMV by recycled RBP. A 1 ml Ni NTA column was pre-loaded with 6xHis-tagged RBP (recovered from supernatant of ultracentrifugation step corresponding to experiment in Figure 7a), followed by introduction of RCNMV (1.5 mg virus at a concentration of 1 mg/ml loaded on the column at 1 ml/min). RCNMV is captured on the column. Upon addition of elution buffer (at ∼ 60 min), RCNMV is co-eluted with RBP. (e) SDS PAGE analysis of RCNMV purified by conventional method (lanes 1, 2, 3) and avidity mediated separation with recycled RBP (lanes 4, 5, 6). Recovery was estimated at nearly 100% by densitometry analysis using pure RCNMV as standard (data not shown). A band is seen below the viral coat protein band in the virus prep purified by conventional method (lanes 1, 2, 3). This is likely a degradation product of RCNMV. (f) Degradation products are also seen in an SDS-PAGE analysis of several months-old RCNMV prep purified using the conventional method (lane 2).
BTPR_1655_sm_SuppFig3.tif7192KSupporting Information Figure 3: SDS-PAGE analysis of elution fractions in avidity mediated separation of RCNMV from plant extract. SDS-PAGE gels for four different independent experiments as indicated by panels (a)-(d). In each case, a Ni-NTA column (1 ml column for panels a, b, c and 5 ml column for panel d) was first saturated with RBP, followed by plant extract loading and a wash step. Wash buffer was 50 mM Tris, 300 mM NaCl, pH 7.5 for (a), 20 mM sodium acetate, pH 5.5 for panel (b), and 20 mM Na2HPO4 for (c) and (d). Finally elution with buffer B (50 mM Tris, 300 mM NaCl, 500 mM imidazole, pH 7.5) was carried out. The extent of impurities in the elution fractions varies with the specific batch of plant extract and/or the buffer used in the wash step. Note that the band immediately below RBP (panels (b), (c), (d)) are due to incomplete denaturation of RBP (denaturation was performed for 5 min at 98° C in a thermocycler) and has been observed in other Sso7d mutants (data not shown). This band is not observed in panel (a) because the samples were boiled for 20 min in water before loading on the gel. Elution fractions in (d) contain the highest levels of impurities and were used in the experiment described in Figure 5.
BTPR_1655_sm_SuppFig4.tif2051KSupporting Information Figure 4: Avidity-mediation separation of RCNMV using a pre-equilibration step between RCNMV and RBP. Chromatograms of avidity mediated capture of RCNMV, wherein RNCMV was equilibrated with RBP (0.02 mg/ml RCNMV in PBS with 0.1% BSA and 2 μM RBP in (a) and 0.002 mg/ml RCNMV in PBS with 0.1% BSA and 1 μM RBP in (b)) for 2 hours on ice prior to loading on a 1 ml (a) or 5 ml Ni-NTA column (b). After a wash step with 50 mM Tris, pH 7.5, 300 mM NaCl, RBP-RCNMV complexes were eluted using elution buffer (50 mM Tris, 300 mM NaCl and 500 mM imidazole, pH 7.5). (c) Recovery of the virus from experiments in (a) and (b) is shown. Fractional recovery is defined as the ratio of the amount of RCNMV obtained in pooled elution fractions to the total amount loaded on to the column. Recovery was calculated by densitometry analysis of elution fractions using pure RCNMV obtained from the conventional method as a reference (d) SDS-PAGE gel of the elution fraction from the chromatogram shown in panel (a). Lanes 2 and 3 correspond to the RCNMV diluted in 0.1% BSA in PBS that was loaded on the 1 ml column and the flow through, respectively. The prominent band in these lanes corresponds to BSA. Lanes 4 through 12 indicate 1 ml fractions collected from 52 through 60 min. Note that the viral coat protein band appears first in lanes 7 and 8, before the RBP band appears, indicating a size-exclusion effect in the column. Also, note that negligible BSA contamination is found in the elution fractions containing RCNMV and RBP.

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