Using this methodology, we were able to purify ~30mg/L of protein complex. The complex was captured using the 6-His tag on Protein C, with all three proteins seen on SDS PAGE after purification. This, in conjunction with analytical size exclusion data, confirmed that we had purified the heterotrimeric complex (Fig.1). After removal of the 6-His tag by TEV protease, a portion of the purified protein complex was then successfully biotinylated through the AviTag sequence on Protein A using biotin ligase.
Fig.1 (a) SDS PAGE analysis of the heterotrimeric complex before and after biotinylation shows the presence and purity of all three proteins. A slight mass shift can be seen for Protein A after biotinylation (b) A-SEC comparison to a standard curve indicates monomeric ABC and ABC-biotin complexes
At Peak Proteins we perform LCMS and MSMS routinely; not only to confirm protein expression, but also to help characterise proteins as part of the QC package. We use a Sciex ExionLC coupled to a Sciex X500B QTOF mass spectrometer with Sciex OS and BioPharmaView Software. The instrument is set up as open access for intact mass measurements for other protein scientists in Peak Proteins, using either a 5 or 10 minute reversed phase method. Having access to protein mass spectrometry within our lab means that we can also employ this as a tool to quickly monitor different stages of the purification and protein production processes in real time, expediting timelines and enabling us to react quickly to our data. In this project, we were able to not only confirm successful co-expression and purification of the heterotrimer but also to demonstrate TEV cleavage of the 6-His tag from Protein C, and the subsequent biotinylation of Protein A. The LCMS data is summarised in Table 1.
(a) All three proteins have the correct mass when adjusting for loss of Met1 (-131 Da) (b) The mass of Protein C is reduced by TEV cleavage to produce a single species of the expected mass (c) After biotinylation, Protein A is shown to have an increased mass of +226 Da, indicating the addition of a single biotin.