Production of Recombinant His-tagged Triple-Flag Peptide in Brevibacillus Choshinensis and Its Utilization As an Easy-to-remove Affinity Peptide.

Triple-FLAG (3 x FLAG)-tagged proteins can be affinity purified through binding to an anti-FLAG antibody and competitive elution with excess free 3 x FLAG peptide. To expand the availability of the 3 x FLAG purification system, we produced a recombinant His-tagged 3 x FLAG peptide in Brevibacillus choshinensis. The screening of connecting linkers between His-tag and the 3 x FLAG peptide, culture containers, and culture media showed that the His-tagged 3 x 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 x FLAG-tagged & alpha;-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 x FLAG peptide can function as an easy-to-remove affinity peptide in the 3 x FLAG purification system.