After extensive analysis of the literature and several rounds of discussion with the client, an appropriate construct was designed which fulfilled the brief regards domain boundaries and the addition of a 6His and Avi-tag to allow biotinylation. Our strategy encompassed the co-expression of the biotin ligase enzyme to allow cellular biotinylation of the Avi-tag.
The supply, and the customer’s final application, permitted the expression and purification to be carried out in the presence of a stabilising ligand, and a suitable detergent, to avoid protein aggregation and the known stability issue. Once these concerns were addressed, purification via affinity capture and a polishing size exclusion step lead to a delivery of milligram quantities of protein. The removal of the detergent was also investigated for the client with a view to supply for other screening techniques but though a second batch of protein was provided it was of much lower yield as the inherent instability returned.