Recombinant wild-type and Ser-to-Ala (S886A and S999A) mutant His-tagged linker proteins spanning Pro683 to Asn1023 of human EPRS were expressed and purified as described7 (link),8 (link). Recombinant active S6K132 (link) and RSK1–3 were from Cell Signaling; Akt1, and Akt2 were from EMD Millipore. Mouse EPRS domains ERS (Met1 (link) to Gln682), linker (Pro683 to Asn1023), and PRS (Leu1024 to Tyr1512) were cloned into pcDNA3 vector with an N-terminus Flag tag using full-length mouse EPRS cDNA (Origene) as template. Flag-tagged mouse wild-type linker and linker with Ser999-to-Ala (S999A) and Ser999-to-Asp (S999D) mutations were generated as described33 (link). Full-length human S6K1 cDNA in pCMV6-Entry vector was purchased from Origene and recloned, deleting the 23-amino acid N-terminus nuclear localization signal, and adding an in-frame upstream 6-His tag and a downstream Myc tag in pcDNA3. Specific Thr389-to-Ala (T389A) and Thr389-to-Glu (T389E) mutations were introduced using primers with the desired mutation and GENEART Site-Directed Mutagenesis System (Invitrogen).
Protocol detail
Purification and Mutagenesis of EPRS Linker
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Corresponding Organization : Cleveland Clinic Lerner College of Medicine
Other organizations : Cedars-Sinai Medical Center, University of Illinois Urbana-Champaign, Institut d'Investigació Biomédica de Bellvitge, Institut Català d'Oncologia, Universitat de Barcelona, University of Cincinnati Medical Center
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Variable analysis
independent variables
- Recombinant wild-type and Ser-to-Ala (S886A and S999A) mutant His-tagged linker proteins spanning Pro^683 to Asn^1023 of human EPRS
- Mouse EPRS domains ERS (Met^1 to Gln^682), linker (Pro^683 to Asn^1023), and PRS (Leu^1024 to Tyr^1512) cloned into pcDNA3 vector with an N-terminus Flag tag
- Flag-tagged mouse wild-type linker and linker with Ser^999-to-Ala (S999A) and Ser^999-to-Asp (S999D) mutations
- Full-length human S6K1 cDNA in pCMV6-Entry vector recloned, deleting the 23-amino acid N-terminus nuclear localization signal, and adding an in-frame upstream 6-His tag and a downstream Myc tag in pcDNA3
- Specific Thr^389-to-Ala (T389A) and Thr^389-to-Glu (T389E) mutations introduced into full-length human S6K1 cDNA
- Not explicitly mentioned
- Not explicitly mentioned
- Recombinant active S6K1
- Recombinant active RSK1-3
- Recombinant active Akt1, and Akt2
- Not explicitly mentioned
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