3.5. PROCEDIMIENTO DE APLICACIÓN DEL HCM
3.5.3. AJUSTE DE LA OFERTA
3.5.3.2. FACTORES DE AJUSTE DE FLUJO DE SATURACIÓN
Small-scale studies have shown that CbiX was expressed well and without excessive proteolysis after induction for approximately 2 hours with IPTG. To facilitate a large scale purification of CbiX, four 750ml LB+A-fC flasks inoculated with AR8882 were incubated overnight at 37°C. Two hours before final harvesting, lOOpM IPTG was added to all cultures.
The bacteria were harvested by centrifugation at 12,500 x g for 15 minutes at 4°C and the pellet resuspended in 20ml of 50mM Tris-HCl (pH7.5) and then sonicated. The soluble
extract was separated by centrifugation at 40,000 x g for 30 minutes at 4°C.
4.2.3.1 Initial Purification Attem pts
After separation of the soluble AR8882 cell extract, several procedures were attempted to isolate the 35kDa band thought to be CbiX. Initial purification attempts employed classical purification methods, thus the extract was subject to differential precipitation with ammonium sulphate. A number of cuts were taken, as indicated in Materials and Methods (2.2.3.4) and the precipitates analysed by SDS-PAGE. Although CbiX precipitated mainly in the 30-50% fraction, no significant purification was observed.
After dialysis of the 30-50% ammonium sulphate precipitate to remove the ammonium sulphate, the extract was applied to an anion exchange chromatography column (DBAE). After application, the column was washed with buffer and the bound protein was eluted by application o f a salt gradient. The 35kDa protein eluted from the column with a sodium chloride concentration of approximately 300mM in 50mM Tris-HCl (pH7.5) but, again, many contaminants were seen when the fractions were analysed by SDS-PAGE.
Fractions from the DEAE column containing CbiX were pooled and concentrated in an Amicon filtration cell, which had been fitted with a PM 10 membrane, to about 10ml. The concentrate was applied to a 150 x 10cm column of Sephacryl S-200. Fractions eluting from the gel filtration column were analysed by SDS-PAGE. This procedure, however, did not appear to purify CbiX significantly.
The m ajor problem throughout these procedures was the apparent loss of the 35kDa protein, presumably by proteolytic breakdown. Thus, although the purification procedures were separating the proteins, there was no net purification due to the on-going degradation of CbiX.
4 .2 .3 .2 N ickel-B in d in g C hrom atography
Tagging proteins with histidine residues has become a fast and efficient method of protein purification [145, 146] as the His tail acts as an efficient metal chelator, facilitating the binding of the protein to a metal affinity column. The 'natural' poly(histidine) m otif of CbiX was used in a similar way to investigate whether it could provide specific binding to the 'His*Bind' affinity column resin (see 2.2.4.4).
The m anufacturer's protocol was followed for preparation and loading o f the metal chelate column, with the exception of the adapted W ash buffer, containing double the manufacture's suggested concentration of NaCl. The soluble fraction from a 3 litre culture
of AR8882 ipKK.cbiX) was applied to the His*Bind column The column was washed with
binding buffer to remove unbound proteins and, after a wash step, the bound material was eluted. The fractions from each step of this purification procedure (unbound material from column loading, washes with Bind and Wash buffers, elution using the Elute buffer, and the final Strip wash), are shown in Figure 4.4.
A large part o f the material from the soluble cell extract of AR8882 did not bind to the nickel-binding column, whereas CbiX and a few other proteins bound quite specifically. On some occasions, a 24kDa protein was also eluted with CbiX, indicating it was binding the column matrix to some extent. Increasing the volume of W ash buffer from 10 to 16 column volumes (usually from 25 to 40ml) was investigated to see if this led to increased
purification (Fig. 4.4). The effect of this was to remove more of the minor contaminants.
However, speed was found to be the most important factor in preparing material for nickel- binding chromatography, as the co-elution of the 24kDa protein was more marked if delays occurred, emphasising the proteolytic sensitivity of CbiX.
An interesting observation was made with respect to the fractions which eluted from the
His*Bind column (H i, Figure 4.4): They were always a red/brown colour. This colour
faded with time either during storage or dialysis at 4°C, with no noticeable effect on the concentration of the 35kDa protein. The exact nature of this coloured material is unknown, but it was specific for strains expressing CbiX.
66
45 36 29 24 20 14 sol 1 2 3 4 5 M H] H2 SFigure 4.4 Purification of CbiX by Nickel Binding Chromatography
The soluble fraction of AR8882 (5ml prepared from a 3 litre culture) was loaded onto a prepared column of H is'B ind matrix. Binding buffer (25ml) and W ash buffer (40ml) were applied before elution of bound material with 5ml 1 x Elute buffer. Eluted material was collected in 2 x 2.5ml fractions and a final Strip wash was performed to remove any remaining material.
The figure shows an SDS-PAGE gel loaded with the following volum es of a 1:1 dilution of sample with disruption buffer:
volume approx. protein
loaded (|al) load (p.g)
sol Soluble cell extract of AR8882 10 100
1 Material from column loading, first 2.5ml 10 100
2 Second 2.5ml collection of unbound material 10 50
3 Bind Buffer wash 10 20
4 Eirst 20ml Wash buffer wash 10 10
5 Second 20ml Wash buffer wash 10 5
Hi Initial 2.5ml Elute fraction 2 25
H2
Second 2.5 ml Elute fraction 2 10s
'Strip' eluate 10 < 54 .2.3.3 D ialysis of Eluted M aterial
The nickel-binding protein (including the 35kDa protein predicted to be CbiX) was eluted from the His*Bind column in a high salt concentration (IM imidazole, 500mM NaCl). This salt was removed by dialysis against a salt-free buffer. The sample was pipetted into dialysis tubing (Sigma, pre-boiled for 30 minutes in ImM EDTA) and dialysed overnight versus 50mM Tris (pH7.5) buffer containing lOOjiM PMSF at 4°C.
4 .2 .3 .4 A nion Exchange C hrom atography
To remove the few contaminating proteins which were observed by SDS-PAGE analysis of the eluted His*Bind fraction, anion exchange chromatography was used. A DEAE Sephacel resin (500ml in a 300 x 30mm column) was equilibrated with 50mM Tris-HCl (pH7.5) and the dialysed sample of CbiX loaded onto it. The column was washed with 50mM Tris-HCl (pH7.5) and the bound protein eluted by application of a 0-500mM NaCl gradient in 50mM Tris-HCl (pH7.5). Fractions (6-8ml) were collected and analysed by SDS-PAGE.
The SDS-PAGE analysis of the eluted fractions from this procedure are shown in Figure
4.5. The 35kDa protein is eluted by a NaCl gradient of approximately 300-350mM in 50mM Tris-HCl (pH7.5). Other proteins, originally present in the eluted fractions from the His*Bind, are either eluted at lower (the majority) or higher (the m inority) NaCl concentrations. Any 24kDa protein remaining from the His»Bind procedure was eluted at approximately 250-300mM - when the material from the His*Bind column had a high content of the 24kDa, there was a significant degree of 'overlap' between the elution of the 35 and 24kDa proteins. It was therefore important to remove as much of the 24kDa protein as possible at the His»Bind stage, using the refined protocol described previously.
The anion exchange procedure, in conjunction with the His*Bind method, thus affords homogeneous protein.
66 45 36 29 24 20 14 P i s ii ii l 58 60 61 62 63 M 64 65 66 67 68 69 70 72
Figure 4.5 Anion Exchange Chromatography of Dialysed Post-His*Bind Material
Post-dialysis pooled His'Bind eluate was applied to a column of DEAE Sephacel, and bound material removed by application of a 0-500mM NaCl gradient in 50mM Tris (pH7.5) buffer.
A total of 112 fractions was collected, and fractions 58-72 are shown in the Figure.
Each fraction corresponds to ~5mM NaCl increments and fractions 58-72 cover the range 240-3 lOmM NaCl.
M is Dalton Mark VII-L marker (Sigma); masses (from top) - 66kDa, 45 kDa, 36kDa, 29kDa, 24kDa, 20kDa and 14kDa.
4 .2 .3 .5 D ialysis and Concentration
A nion exchange fractions containing CbiX were pooled and the preparation was concentrated from approximately 80ml to 5ml using an Amicon ultrafiltration unit fitted with a PM 10 membrane under nitrogen at a pressure of 2 bar (30 Ibf/in^, 200kPa). The resultant sample was dialysed overnight at 4°C against lOmM Tris (pH7.5).
4 .2 .3 .6 F reeze-D ry in g
The purified protein was frozen rapidly in liquid nitrogen and then lyophilised and stored at -20°C.