A novel protein purification strategy mediated by the combination of CipA and Ssp DnaB intein.

Protein purification is an indispensable step in diverse fields of biological research or production process. Conventional purification methods including the affinity purification or the usage of self-aggregating tags suffered from many drawbacks such as the complicated steps, high cost and low efficiency. Moreover, the fusion tag usually had negative effects on the activity of the target protein. To address the above issues, here we propose a novel protein purification method which needs simple operation steps, and this method is mediated by the combination of CipA protein and a mini-intein (Synechocystis sp. PCC6803 DnaB, Ssp DnaB), depending on the assembly function of CipA and the self-cleavage function of Ssp DnaB. To realize the purification, CipA-DnaB-eGFP protein was expressed and assembled into protein crystalline inclusions (PCIs) in E. coli. Then, only cell lysis, cleavage and centrifugation steps were required to purify eGFP. Purified eGFP was in the supernatant with a purity of over 90%. The cleavage efficiency and the yield of eGFP reached 51.96% and 13.99 ± 0.88 mg/L fermentation broth, respectively. Furthermore, to broaden the application of this approach, three other proteins which were maltose binding protein (MBP), ketoisovalerate decarboxylase (Kivd) and alcohol dehydrogenase (AdhP) were purified with high cleavage efficiency. The purified Kivd and AdhP remained high specific activities. This work demonstrated an effective and convenient protein purification method.