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

Bacteriophytochromes are phytochrome-like light-sensing photoreceptors that use biliverdin as a chromophore. To study the biochemical properties of the Deinococcus radiodurans bacteriophytochrome (DrBphP) protein, two anti-DrBphP mouse monoclonal antibodies (2B8 and 3H7) were generated. Their specific epitopes were identified in our previous report. We present here fine epitope mapping of these two antibodies by using truncation and substitution of original epitope sequences in order to identify minimized epitope peptides. The previously reported original epitope sequences for 2B8 and 3H7 were truncated from both sides. Our analysis showed that the minimal peptide sequence lengths for 2B8 and 3H7 antibodies were nine amino acids (RDPLPFFPP) and six amino acids (PGEIEE), respectively. We further characterized these peptides in order to investigate their reactivity after single deletion and single substitution of the original peptides. We found that single-substituted 2B8 epitope (RDPLPAFPP) and dual-substituted 3H7 epitope (PGEIAD) showed significantly increased reactivity. These two antibodies with high reactivity for the short modified peptide sequences are valueble for developing new peptide tags for protein research.

Epitope mapping of monoclonal antibodies for the Deinococcus radiodurans bacteriophytochome.

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.

Designing antioxidant peptides.

BACKGROUND: Ionizing radiation causes the generation of damaging reactive oxygen species that lead to cellular damage and death. Organisms such as Deinococcus radiodurans have evolved mechanisms for extreme resistance to ionizing radiation, and resistance has been shown to be a consequence of protection of critical proteins from oxidative inactivation. OBJECTIVES: D. radiodurans accumulates high levels of manganese and of small peptides that together are protective. Our aim was to rationally design antioxidant peptides. METHODS: Amino acid analysis was utilized to determine the rates of loss of the 20 amino acids exposed to varying doses of irradiation. The activity of glutamine synthetase and methionine sulfoxide reductase was assayed to follow their inactivation by irradiation. RESULTS: The ability of an amino acid to protect enzymes from inactivation by ionizing radiation paralleled its sensitivity to ionizing radiation. Based on this observation and the ability of histidine to confer water solubility, we synthesized the hexapeptide His-Met-His-Met-His-Met and found that it provided markedly increased protection against irradiation. DISCUSSION: Small peptides containing histidine and methionine were readily soluble and provided enzymes with remarkable protection from inactivation by ionizing radiation.