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a charged resin. As the protein solution flows through the column, proteins bind to the resin on the basis of their net charge. We used cation exchange chromatography, where the resin is negatively charged and so binds proteins with positive net charge. The stronger the net positive charge, the stronger the protein binds to the resin. Once the sample has passed through the column, a steady gradient of increasing

NaCl concentration was applied. The Na+ions displace the proteins, binding to the

negatively charged resin more strongly. The first proteins to be displaced are those which have the least affinity for the column resin. Proteins are therefore eluted from the column in order of increasing positive net charge. The material eluted from the column is collected in small volumes (fractions) so that the proteins are separated. As the material is eluted, the presence of protein is detected by measuring

absorbance at 280 nm (A280).

Initially, 20 mM tris(hydroxymethyl)aminomethane (TRIS) buffer at pH =

7.8 was used during the expression and purification [Liu et al., 1990]. The isoelectric

point of CypA — the pH value at which the net charge is zero — is estimated to

be 7.68 [ProtParam tool, 2013]. At pH = 7.8 therefore, CypA has a small net

negative charge. We therefore tried using an anion exchange column. Under these conditions, a lot of protein was lost during ion exchange due to most of the CypA not binding to the column. In response, the pH of the system was lowered to below 7.68 and cation exchange was used. At lower pH we observed improved binding of CypA to the ion exchange column and hence a better yield during purification. In order to lower the pH further from the isoelectric point the buffer system was changed to HEPES which is more effective at lower pH than TRIS. Although some material was still lost by not binding to the ion exchange column, the yield became large enough for us to continue. The pH was not lowered further since yields were satisfactory

and CypA remained stable in HEPES at pH = 6.5. We used a 10 mL Source 30S

column (from GE Healthcare) containing a resin with a negative charge for cation

exchange. The column was connected to an ¨AKTA purifier 100 fast protein liquid

chromatography (FPLC) system (from GE Healthcare). The column, which was stored in ethanol, was first equilibrated in 20 mM HEPES buffer, pH 6.5. This was done by running at least 30 mL (three column volumes) of buffer through the column. The protein sample was loaded onto the column using an injection loop. A gradient from 0 –100 % of a second buffer of 20 mM HEPES with 150 mM NaCl

at pH 6.5 was added over 40 minutes. The buffers were run through the ¨AKTA

purifier at 5.0 mL/min, and so a total of 200 mL was passed through the column — equivalent to 20 column volumes. Eluted fractions were collected in volumes of 2.5 mL throughout, with protein elution monitored using UV absorbance at 280 nm

75 70 80 85 90 95 100 105 110 400 300 200 100 0 A280 ( mAU ) Volume (mL)

Figure 5.3: An ion exchange chromatogram showing elution of CypA. The A280 in

mAU, shown in blue, is recorded as material is eluted from the column. The space between two red dashes indicates one 2.5 mL fraction. The green line represents the gradient of [NaCl], which increases from 0.0 mM at 70 mL to 18.75 mM at 95 mL.

(A280). Figure 5.3 shows an example of an ion exchange chromatogram for CypA.

The blue absorbance peak shows where the majority of protein has been eluted from the column.

The composition of the fractions corresponding to large A280peaks in the ion

exchange chromatogram were analysed using SDS-PAGE. Figure 5.4 is an example of such an SDS-PAGE gel. These samples are far purer than the sample before ion exchange (Figure 5.2), yet there remains a protein of higher molecular weight in the sample along with CypA. The bands in the gel corresponding to this unwanted protein are labelled with an X in Figure 5.4. The columns labelled B in Figure 5.4 show the protein in the sample which passed through the ion exchange column without binding to the resin. Clearly there is some CypA which has not bound to the column. This could be because of the column is overloaded and so some CypA cannot bind. The affinity of CypA for the column is not particularly strong, since CypA is eluted at a low concentration of NaCl. To counteract this problem, the sample was split in half prior to loading and then ran two IEC experiments were run in sequence — one for each half of the protein sample. This appeared to decrease the relative abundance of CypA in the fractions which did not bind to the columns (data not shown). There was not a notable increase in final protein yield, however, and the problem of some protein not binding to the column still remained. Since IEC separates proteins according to their net overall charge, it is possible for

Figure 5.4: An SDS-PAGE gel showing incomplete purification as a result of IEC. Column A is the low molecular weight marker. The mass in kDa for each protein component of the marker is labelled. The columns B show the material which did not bind to the column. Columns C are selected fractions following an IEC purification step. The bands corresponding to CypA are labelled. The bands for the unwanted higher molecular weight protein are labelled with an X.

two or more different proteins with different masses to be eluted from the column at the same time. The SDS-PAGE gel in Figure 5.4 demonstrates that this has indeed happened. Nevertheless, as shown by the columns labelled C in Figure 5.4, IEC succeeded isolating CypA from most of the other proteins in the initial sample.