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Recombinant Antibody Expression> | … The lysate was incubated with anti-DYKDDDDK G1 affinity beads (Genscript) for 3 h at 4 C and washed with 20 column volumes of purification buffer. The recombinant protein was eluted using 20 column columns purification buffer supplemented with 1 μM DYKDDDDK FLAG … | Get A Quote |
Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-Å resolution X-ray crystal structure of a TSEN15-34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant ... More