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Updated: Jul 26, 2025

Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes
Published on: October 3, 2019
Production of recombinant His-tagged triple-FLAG peptide in Brevibacillus choshinensis and its utilization as an
Daiki Sugihara1, Fuka Ono2, Motoki Sugino2
1United Graduate School of Agricultural Science, Gifu University, Tokai National Higher Education and Research System, Gifu, Japan.
Abstract:
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.

