Epitope mapping of monoclonal antibodies for the Deinococcus radiodurans bacteriophytochome

Abstract

Bacteriophytochromes (BphP) are phytochrome-like light sensing proteins in bacteria, which use biliverdin as a chromophore. In order to study the biochemical properties of the DrBphP protein, five (2B8, 2C11, 3B2, 3D2, and 3H7) anti-DrBphP monoclonal antibodies were produced through the immunization of mice with purified full-length DrBphP and DrBphN (1-321 amino acid) proteins, and epitope mapping was then carried out. Among the five antibodies, 2B8 and 2C11 preferentially recognized the N-terminal region of BphP whereas 3B2, 3D2, and 3H7 showed preference for the C-terminal region. We performed further epitope mapping using recombinant truncated BphP proteins to narrow down their target sequences. The results demonstrated that each of the five monoclonal antibodies recognized different regions on the DrBphP protein. Additionally, epitopes of 2B8 and 3H7 antibodies were discovered to be shorter than 10 amino acids (2B8: RDPLPFFPP, 3H7: PGEIEEA). These two antibodies with such specific recognition epitopes could be especially valuable for developing new peptide tags for protein detection and purification.

Arabidopsis RING E3 ubiquitin ligase JUL1 participates in ABA‐mediated microtubule depolymerization, stomatal closure, and tolerance response to drought stress

“…The C-terminal RING domain (amino acids 428-525) of JUL1 (RING ) and the single amino acid substitution RING C493S were tagged with the 2B8 epitope (RDPLPFFPP) (Kim et al, 2014). The 2B8-RING 428-525 and 2B8-RING C493S constructs were transformed into Escherichia coli BL21 cells, and the expressed MBP-2B8-RING 428-525 and MBP-2B8-RING C493S recombinant proteins (300 ng) were subjected to in vitro self-ubiquitination assay as described by Cho et al (2008).…”

Fine Mutational Analysis of 2B8 and 3H7 Tag Epitopes with Corresponding Specific Monoclonal Antibodies

“…In this study, we characterized and analyzed the epitope sequences for mAb 2B8 and 3H7 using Western blot and indirect ELISA. In a previous study, we characterized the original epitopes for mAb 2B8 and 3H7, which are short linear peptides ( Kim et al, 2014 ). The specific amino acid sequences of both epitopes were unique compared with the amino acid sequences of known proteins.…”

“…In our previous report, 2B8 and 3H7 peptide tags were identified as parts of a D. radiodurans bacteriophytochrome (DrB-phP), the phytochrome-like photoreceptor designated as bacteriophytochromes ( Bhoo et al, 2001 ; Davis et al, 1999 ). We originally generated five DrBphP-specific monoclonal antibodies, including 2B8 and 3H7, and mapped their epitope positions for use in structural and biochemical studies of bacteriophytochromes ( Kim et al, 2014 ). In the present study, we performed mutational mapping including internal deletion and internal substitution of two epitope tag peptides reacted with 2B8 and 3H7 antibodies for ELISA and Western blot analysis.…”