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Purification of both the untagged recombinant NS7 and VPg was carried out as described in Section 2.10.2 and 2.10.3. Briefly, cells were harvested and lysed.

The lysates were then subjected to nucleic acid precipitation using PEI, ammonium sulphate (AS) precipitation to precipitate the protein, dialysis and further subjected to several rounds of column chromatography (Figure 4.11a).

As shown in Figure 14.11b, the untagged recombinant NS7 and VPg were successfully purified after being isolated via several different columns during the chromatography process. Compared to the his-tagged protein purification, during which the fusion protein binds to a nickel-nitriloacetic acid (Ni-NTA) column and can be readily eluted with imidazole, the purification of the non-fusion proteins was more complex. After removal of nucleic acid and subsequent dialysis, the protein of interest was purified by chromatography on phosphocellulose, heparin and SP sepharose resins. Due to the untagged nature of the protein, traditional purification approaches were used whereby proteins bound to various resins were eluted using a salt

(a) (b)

Figure 4.11 Purification of non-his MNV NS7 and VPg. (a) Flow chart of the protein preparation process starting from cell lysis until column chromatography using AKTAprime™ Automated Liquid Chromatography System from GE Healthcare. (b) Samples from the NS7 and VPg preparation were run on 12.5% and 15% SDS PAGE respectively and visualised by comassie blue stain. (S1) = total lysate, (S2) = soluble protein, (S3) = supernatant from protein precipitation, (S4) = precipitated protein by ammonium sulphate, (S5) = post dialysis of precipitated protein, (S6) = Final purified protein after chromatography and dialysis.

gradient and the cleanest fractions pooled prior to moving to the next step. The optimised procedures are described below.

4.2.2.2.1 Purification of the untagged recombinant MNV NS7

The protocol for column chromatography purification of the untagged NS7 is described in further detail in Section 2.10.2. Briefly, after the removal of nucleic acid and an initial ammonium sulphate mediated precipitation and dialysis, samples of untagged NS7 in Buffer A+100 mM NaCl were loaded onto a Phosphocellulose (PC) column, equilibrated in the same buffer, and the gradient eluted with 100 mM to 1M NaCl Buffer A. The untagged NS7 was found not to bind to the column under the conditions used (data not shown). However by including a PC column separation, many nucleases and other nucleic acid binding proteins can be removed along with some contaminating proteins. The collected pass through from the PC column was further loaded onto a heparin column and the untagged NS7 was eluted at around 400 mM NaCl (Figure 4.12a). Heparin is a mucopolysaccharide, which contains a repeating dimer of hexuronic acid and D-glucosamine. The choice to use this column to purify the untagged NS7 was made due its well-known interaction with nucleic acid-binding proteins. The cleanest fractions were then pooled, diluted in Buffer A to the desired final concentration of NaCl (typically 40-100 mM) and subjected to further purification by loading onto a SP Sepharose column. The untagged NS7 protein bound weakly to the SP column but it provided a useful resin with which to remove substantial contaminants. The untagged NS7 eluted from the SP resin at around 40 mM NaCl (Figure 4.12b). The cleanest fractions were pooled together again, diluted with Buffer A and loaded onto heparin columns again to concentrate the sample and remove additional contaminating proteins. The untagged NS7 was eluted from the column at between 300 and 400 mM NaCl (Figure 4.12c). Finally, the cleanest fractions were pooled together, dialysed and concentrated as described in Section 2.10.2 The final concentrated untagged NS7 sample (S6) was resolved on a 12.5%

SDS PAGE. (Figure 4.11b top panel).

(a)

(b)

(c)

Figure 4.12 Series of column chromatography purifications for the untagged MNV NS7. (a) Chromatography purification of NS7 using the heparin column. The pass through NS7 samples from the PC column were collected and loaded onto the heparin column and gradient eluted with NaCl containing buffer as described in Section 2.10.2.

Fractions 14-18 were pooled together and diluted with a buffer solution without NaCl. (b) Chromatography purification of NS7 using the SP column. The diluted pooled fractions from the heparin column were loaded onto the SP column. Gradient elution was performed as described in Section 2.10.2. Fractions 15-30 were pooled together and diluted with buffer A with no NaCl and NP-40 (c) Final chromatography purification of NS7 using the heparin column. The diluted pooled fractions from the SP column were loaded onto the heparin column again and gradient eluted as described in Section 2.10.2.

Fractions 15-18 were pooled together, dialysed against NaCl containing buffer, concentrated down using a spin column  and subjected to concentration determination as described in Section 2.10.2. All protein samples were resolved on 12.5% SDS PAGE and visualised using coomassie blue stain. M= protein marker in kDa, L= loaded sample, P=

pass through sample.

4.2.2.2.2 Purification of the untagged recombinant MNV VPg

The detailed procedure of column chromatography purification of the untagged VPg is described in Section 2.10.3. Briefly, after the removal of nucleic acid, ammonium sulphate precipitation and dialysis, the sample was loaded onto PC column previously equilibrated with 250 mM NaCl Buffer A. Gradient elution was performed with 250 mM to 1.5 M NaCl buffer A and VPg was found to elute between 400 to 500 mM NaCl (Figure 4.13a). The cleanest fractions were pooled together and diluted with Buffer A to a final concentration of around 100 mM NaCl. The SP resin was employed as a further ‘untagged VPg purification column’, the sample being loaded onto the SP column and gradient eluted at around 600 mM NaCl Buffer A

(a)

(b)

Figure 4.13 Series of column chromatography purifications for the untagged recombinant MNV VPg. (a) Chromatography purification of VPg using the PC column.

The dialysed sample was loaded onto the PC column and gradient eluted with NaCl containing buffer as described in Section 2.10.3. Fractions 14-18 were pooled together and diluted with buffer solution without NaCl. (b) Chromatography purification of VPg using the SP column. The diluted pooled fractions from the PC column were loaded onto the SP column and gradient eluted with NaCl containing buffer A as described in Section 2.10.3.  Fractions 33-39 were pooled together and dialysed against NaCl containing buffer, concentrated down using a spin column  and subjected to concentration determination as described in Section 2.10.3. All protein samples were resolved on 15% SDS PAGE and visualised using Coomassie Blue stain. M= protein marker in kDa, L= loaded sample, P=

pass through sample. M= protein marker in kDa, L= loaded sample, P= pass through sample.

(Figure 4.13b). The cleanest fractions were pooled together, dialysed and concentrated down using a spin column as described in Section 2.10.3. The final purified and concentrated product of the untagged VPg sample (S6) was resolved on a 15% SDS PAGE. (Figure 4.11b bottom panel).

4.2.3 Characterisation of the purified untagged recombinant MNV NS7