Purification with Talon TM affinity chromatography

With affinity chromatography, many factors influence the quantity and quality of purification, such as the scale of crude extract including expressed proteins, the optimal environment for isolating and stabilizing the objective protein (salt concentration, pH etc). With the ion exchange chromatography, 300 mM NaCl was used in all solutions during the purification. Assays to test optimal pH and whether glycerol is helpful were performed as described below.

Briefly, the cytoplasmic fraction isolated from E. coli ΔtatABCDE cells was applied to an equilibrated 2.5 ml TalonTM affinity column that specifically bound to his- tagged YedY. The column was washed using equilibration buffer to remove unbound protein, and eluted with equilibration buffer supplemented with imidazole. Protein presented in column fractions was resolved on SDS-PAGE, and the gel was immunoblotted with anti-his antibody or stained by silver stain.

Figure 4.2.3 shows the purification performed at pH 8.0. The anti-his immunoblot has shown that not all YedY bound to the column and protein was detected in the flow-through (FT) as well as wash fractions. YedYhis that did bind specifically eluted from the column with a clear peak in the elution 4 and 5. However, some small proteolytic clipping of the protein was also found in two elutions. In terms of silver stain of eluted fractions, higher molecular weight bands were present which were absent in the immunoblot implying the presence of unspecific proteins co-eluted from the column. In short, the environment of pH 8.0 is not suitable to purify and stabilize YedY using a Talon affinity column.

0 100 200 300 400 500 Co n ce n tr ation (u g/ m l) YedY E1 E2 E3 E4 E5 E6 E7

Figure 4.2.3 Talon affinity chromatography of pre-YedY with pH 8.0 YedY was amplified with C-terminal his-tag into arabinose inducible pBAD24. It started the 3L culture to over-express YedY in ∆tatABCDE cells. E. coli cytoplasmic fraction (C) was isolated from cells and directly applied to Talon affinity column. The flow-through (FT) sample collected and unbound proteins were washed (W). Bound proteins were eluted by elution buffer. All elution fractions (E) were measured by Bradford assay and analysed by silver stain as well as immunoblot with anti-his antibody.

The purification carried out with solutions at pH 5.0 is shown in Figure 4.2.4. The immunoblot shows that some YedY was detected in the flow-through (FT) indicating protein that did not bind to the column effectively. YedYhis bound was then specifically eluted from the column and seemed to be very pure in the immunoblot. However, there is still some contaminations when purity was assessed by silver stain of SDS-PAGE. Moreover, the quantity of relatively pure YedY was limited (based on the BCA assay). Thus, pH 5.0 can improve the purity of protein, but fails to increase the amount of protein. In order to get enough purest protein to analyze the structure, more conditions need to be changed and examined such as adding glycerol in buffers which was suggested to remove unspecific proteins and improve the purity.

0 20 40 60 80 100 Co n ce n tr ation (u g/ m l) YedY E1 E2 E3 E4 E5

Figure 4.2.4 Talon affinity chromatography of pre-YedY with pH 5.0 YedY was amplified with C-terminal his-tag into arabinose inducible pBAD24. It started the 1L culture to over-express YedY in ∆tatABCDE cells. E. coli cytoplasmic fraction (C) was isolated from cells and directly applied to Talon affinity column. The flow-through (FT) sample collected and unbound proteins were washed (W). Bound proteins were eluted by elution buffer. All elution fractions (E) were measured by Bradford assay and analysed by silver stain as well as immunoblot with anti-his antibody.

Based on the experiment carried out at pH 5.0, 5% glycerol was added to help the purification. The immunoblot in Figure 4.2.5 shows that some YedY was lost in the flow-through (FT) indicating protein that still was not effectively bound to the column. Importantly, the specifically bound YedYhis was eluted in one elution. It was shown that this YedY was pure, even when concentrated to a small volume, but a higher molecular weight band after concentration was present in the silver stain of SDS-PAGE. The purification of YedY was not improved by adding glycerol, because the quantity of protein was still a key problem to solve. Hence, it was important to increase the binding efficiency of protein using the affinity chromatography.

Figure 4.2.5 Talon affinity chromatography of pre-YedY with pH 5.0 as well as adding of Glycerol YedY was amplified with C-terminal his-tag into arabinose inducible pBAD24. It started the 0.5L culture to over-express YedY in ∆tatABCDE cells. E.coli cytoplasmic fraction (C) was isolated from cells and directly applied to Talon affinity column. The flow-through (FT) sample collected and unbound proteins were washed (W). Bound proteins were eluted by elution buffer. Elution fractions (E) and concentrated elution fraction (CE) were measured by Bradford assay and analysed by silver stain as well as immunoblot with anti-his antibody.

4.2.2.3 Purification with Immobilized metal ion affinity chromatography