Using EDTA as a Strategy to Reduce Protein Aggregation

A client requested the large-scale production of a protein that had previously been optimised for purification during feasibility studies. This protein had proven tricky to purify during the feasibility stage and two attempts had been taken to establish a suitable purification strategy. High protein concentrations were found to be compromising the stability of the protein, so sample volumes were adjusted to minimise the risk. The buffer and pH conditions had also been optimised and the large-scale purification attempt was to be scaled up accordingly, to maximise soluble protein yield.

The problem

The large scale purification attempt ran into an issue at the elution stage, when the protein, upon being eluted off the Ni-NTA affinity column ,was found to be highly prone to aggregating. The eluted protein was displaying the tell-tale, cloudy appearance in the collection tube. After attempts to increasing the elution volumes and dilute the protein, the aggregation issue was somewhat reduced, however the soluble protein yield was still impacted due to aggregation loss.

The solution

EDTA (Ethylenediaminetetraacetic acid) is a chelating agent that can bind and chelate various metals including nickel. During purification, nickel can leach from the Ni-NTA column and end up in the washes and elutions collected. The thinking here is that at high protein concentrations, the stripped nickel can increase protein-protein self-interactions via the His-tag, increasing the risk of aggregates forming. Theoretically, using EDTA to chelate the stripped nickel could help to mitigate this problem. However, EDTA could not be added to the affinity column as it will chelate the nickel, thereby reducing the amount of protein binding to the column.

A solution was tested whereby a precalculated amount of EDTA was added to the collection tubing. The elution was collected into the buffer containing EDTA and the EDTA would chelate any stripped nickel ions from the column and decrease protein-protein self-interactions, thereby reducing the risk of aggregation. In a direct comparison, where the only change in experimental strategy was the introduction of EDTA in the collection buffer, visible aggregation was reduced by more than 80%. The trace below shows the protein after size exclusion chromatography had been performed and we managed to achieve yields of 20mg/L of >95% pure protein! This strategy has also worked for us successfully for various other proteins. The EDTA is removed via dialysis further downstream and had no effects on activity of the protein in assays. It’s a neat little ‘cheat’ trick that works rather well although we recommend not to use it with any metalloenzymes!