Production of Recombinant His-tagged 3× FLAG Peptide in Brevibacillus choshinensis and its Utilization as an Easy-to-Remove Affinity Peptide.

Triple-FLAG (3× FLAG)-tagged proteins can be affinity purified through binding to an anti-FLAG antibody and competitive elution with excess free 3× FLAG peptide. To expand the availability of the 3× FLAG purification system, we produced a recombinant His-tagged 3× FLAG peptide in Brevibacillus choshinensis. The screening of connecting linkers between His-tag and the 3× FLAG peptide, culture containers, and culture media showed that the His-tagged 3×FLAG peptide with an LA linker was most expressed in 2SY medium using a baffled shake flask. The peptide was affinity-purified to give a yield of about 25 mg/L of culture. The peptide was effective for eluting 3× FLAG-tagged α-amylase from anti-FLAG magnetic beads. Finally, the peptide remaining in the amylase fraction was removed by His-tag affinity purification. These results show that the recombinant His-tagged 3× FLAG peptide can function as an easy-to-remove affinity peptide in the 3× FLAG purification system.