pcr amplified rpl10a coding sequence  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: Exploration of the proxiOME of large subunit ribosomal proteins reveals Acl1 and Bcl1 as cooperating dedicated chaperones of Rpl1

doi: 10.1093/nar/gkag264

Figure Lengend Snippet: The conserved Bcl1 interacts via its WD40 β-propeller with Rpl1. ( A ) Proximity labelling assay with C-terminally miniTurbo-tagged RPL10A (HsRPL10A-miniTurbo) in HeLa cells. The RPL10A bait r-protein and selected enriched proteins are written in bold, the red dot highlights the highly enriched WDR89. ( B ) AlphaFold3 model of the Bcl1–Rpl1 complex. The seven-bladed β-propeller domain of Bcl1 is coloured in green and the C-terminal extension in light green; the position of residue Asn322 (N322) is indicated to better visualize from where the C-terminal extension emanates. ( C ) AlphaFold3 model of the WDR89–RPL10A complex (left) and its structural superposition with the AlphaFold3 model of the Bcl1–Rpl1 complex (right). ( D ) Predicted electrostatic surface potential of Bcl1 (left) and close-up view of two of the three Rpl1 sites, indicating residues predicted to form H-bonds with Bcl1, that are in contact with the negatively charged top surface of the β-propeller (right). ( E–G ) Y2H interaction assays between the full-length Rpl1 and Bcl1 proteins ( E ), between full-length Rpl1 and the C-terminally truncated Bcl1.N366 and Bcl1.N325 variants or the C-terminal extension of Bcl1 (323C) ( F ), and between the indicated Rpl1 mutant variants and either Bcl1 or Acl1 ( G ). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; E, Glu; K, Lys; R, Arg. ( H ) In vitro binding assay between Bcl1.N366 and Rpl1. Bcl1.N366-(His) 6 or Bcl1.N366 and Rpl1b were co-expressed in E. coli and purified by Ni–NTA affinity purification. Proteins were revealed by SDS–PAGE and Coomassie staining (top) or by western blotting using anti-His and anti-Rpl1 antibodies (bottom). Bands corresponding to Bcl1.N366-(His) 6 and Bcl1.N366 or to Rpl1b are indicated by blue or black arrowheads.

Article Snippet: The DNA sequence coding for the Homo sapiens RPL10A protein was PCR-amplified from plasmid pADH111-HsRPL10A (pDK10427), generated by cloning the PCR-amplified RPL10A coding sequence [template pNTI194 (Addgene plasmid #84266)] into the Nde I/ Bam HI-restricted plasmid pADH111-LTV1 (pDK3331), and cloned by Gibson assembly between the Nhe I and Pst I restriction sites of the lentiviral donor vector pSKP-32, a pCW57.1-derived plasmid bearing the MND-Blasticidin resistance cassette instead of the hPGK-puromycin resistance cassette [ ], to generate plasmid pDS79 containing the RPL10A gene under the transcriptional control of a doxycycline-inducible promoter and fused at its 3′ end to sequences encoding the V5 tag, the miniTurbo (MT) biotin ligase, and the HA tag.

Techniques: Residue, Mutagenesis, In Vitro, Binding Assay, Purification, Affinity Purification, SDS Page, Staining, Western Blot