A phosphorylation pattern-recognizing antibody specifically reacts to RNA polymerase II bound to exons

Abstract

The C-terminal domain of RNA polymerase II is an unusual series of repeated residues appended to the C-terminus of the largest subunit and serves as a flexible binding scaffold for numerous nuclear factors. The binding of these factors is determined by the phosphorylation patterns on the repeats in the domain. In this study, we generated a synthetic antibody library by replacing the third heavy chain complementarity-determining region of an anti-HER2 (human epidermal growth factor receptor 2) antibody (trastuzumab) with artificial sequences of 7–18 amino-acid residues. From this library, antibodies were selected that were specific to serine phosphopeptides that represent typical phosphorylation patterns on the functional unit (YSPTSPS)2 of the RNA polymerase II C-terminal domain (CTD). Antibody clones pCTD-1stS2 and pCTD-2ndS2 showed specificity for peptides with phosphoserine at the second residues of the first or second heptamer repeat, respectively. Additional clones specifically reacted to peptides with phosphoserine at the fifth serine of the first repeat (pCTD-1stS5), the seventh residue of the first repeat and fifth residue of the second repeat (pCTD-S7S5) or the seventh residue of either the first or second repeat (pCTD-S7). All of these antibody clones successfully reacted to RNA polymerase II in immunoblot analysis. Interestingly, pCTD-2ndS2 precipitated predominately RNA polymerase II from the exonic regions of genes in genome-wide chromatin immunoprecipitation sequencing analysis, which suggests that the phosphoserine at the second residue of the second repeat of the functional unit (YSPTSPS)2 is a mediator of exon definition.

S100A4 released from highly bone-metastatic breast cancer cells plays a critical role in osteolysis

“…The scFv-displaying phages were subjected to phage enzyme immunoassays, as described previously. 43 Plasmid DNA was prepared from selected clones that had binding activity for human rS100A4. The sequence of the V H and V L chains of the scFv Ab was identified.…”

Next-generation sequencing enables the discovery of more diverse positive clones from a phage-displayed antibody library

“…14 Four rounds of bio-panning were performed to screen scFv clones from the library following a previously reported procedure. 15 For each round of bio-panning, 5 × 10 6 magnetic beads (Dynabeads M-270 epoxy) (Invitrogen) coated with 1.5 μg recombinant PSA protein were used.…”

Optimized monoclonal antibody treatment against ELTD1 for GBM in a G55 xenograft mouse model

“…Phage clones showing cross reactivity against human and mouse ELTD1 were selected, and their nucleotide sequences were determined by Sanger sequencing. The gene of selected scFv clone was subcloned into a modified mammalian expression vector encoding the hinge region of human IgG1 and the CH2‐CH3 domains of rabbit IgG at the 3′ region as reported previously . The expression vectors encoding anti‐ELTD1 scFv‐rFc fusion were transfected into HEK293F cells (Invitrogen) as described above.…”

“…A phage-displayed chicken single-chain variable fragment (scFv) library was constructed using total RNA isolated from the bone marrow, spleen and bursa of Fabricius of immunized chickens, as described previously. 19 The expression vectors encoding anti-ELTD1 scFv-rFc fusion were transfected into HEK293F cells (Invitrogen) as described above. 18 Phage clones showing cross reactivity against human and mouse ELTD1 were selected, and their nucleotide sequences were determined by Sanger sequencing.…”

“…The gene of selected scFv clone was subcloned into a modified mammalian expression vector encoding the hinge region of human IgG1 and the CH2-CH3 domains of rabbit IgG at the 3′ region as reported previously. 19 The expression vectors encoding anti-ELTD1 scFv-rFc fusion were transfected into HEK293F cells (Invitrogen) as described above. The scFv-rFc fusion protein was purified from the culture supernatants of transiently transfected HEK293F cells using protein A Sepharose column (Repligen) according to the manufacturer's instructions.…”