Production of recombinant His-tagged triple-FLAG peptide in <i>Brevibacillus choshinensis</i> and its utilization as an easy-to-remove affinity peptide-Reference-Cited by-同舟云学术

Production of recombinant His-tagged triple-FLAG peptide in Brevibacillus choshinensis and its utilization as an easy-to-remove affinity peptide

Published:2023-06-16 Issue:9 Volume:87 Page:1029-1035
ISSN:1347-6947
Container-title:Bioscience, Biotechnology, and Biochemistry
language:en
Short-container-title:

Author:

Sugihara Daiki¹,Ono Fuka²,Sugino Motoki²,Suzuki Hiromi²,Endo Noriko³,Shimada Atsuhiro³,Ebihara Akio³⁴⁵^ORCID

Affiliation:

1. United Graduate School of Agricultural Science, Gifu University, Tokai National Higher Education and Research System , Gifu , Japan

2. Graduate School of Natural Science and Technology, Gifu University, Tokai National Higher Education and Research System , Gifu , Japan

3. Faculty of Applied Biological Sciences, Gifu University, Tokai National Higher Education and Research System , Gifu , Japan

4. Preemptive Food Research Center (PFRC), Gifu University Institute for Advanced Study , Gifu , Japan

5. Department of Chemical Engineering, Indian Institute of Technology Guwahati , Guwahati, Assam , India

Abstract

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.

Funder

JSPS

Publisher

Oxford University Press (OUP)

Subject

Organic Chemistry,Molecular Biology,Applied Microbiology and Biotechnology,General Medicine,Biochemistry,Analytical Chemistry,Biotechnology

Link

https://academic.oup.com/bbb/advance-article-pdf/doi/10.1093/bbb/zbad079/50836944/zbad079.pdf

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