Recombinant Expression of TIA-1 RRM2,3 Protein

To construct the plasmid for expression of TIA-1 RRM2,3 (93-274), pGEX-4T-2 (GE Healthcare) was modified to bear a 6xHis tag and a thrombin cleavage site. TIA-1 RRM2,3 was inserted directly after the thrombin site using oligonucleotides TIA-1 RRM2,3-For (GCGTGGATCCCCAGGAATTCCCAATCATTTCCATGTCTTTGTTGGTG) and TIA-1 RRM2,3-rev (CAGTCACGATGCGGCCGCTTATTTGCCCCAATAGCATTTCACAAC). To construct the plasmid TIA-1 RRM2,3 bearing mutations H94A, H96 A and H94A H96A, site-directed mutagenesis by inverse PCR (Liu and Naismith, 2008 (link)) was performed using the oligonucleotide pairs:
TIA-1 RRM2,3-H94A-For: CCAATgctTTCCATGTCTTTGTTGGTGATCTCAGCCCAG
TIA-1 RRM2,3-H94A-Rev: ATGGAAagcATTGGGAATTCCTGGGGATCCACG
TIA-1 RRM2,3-H96A-For: TTTCgctGTCTTTGTTGGTGATCTCAGCCCAG
TIA-1 RRM2,3-H96A-Rev: AAAGACagcGAAATGATTGGGAATTCCTGGGGATCCACG
TIA-1 RRM2,3-H94A H96A-For: CCAATgctTTCgcTGTCTTTGTTGGTGATCTCAGCCCAG
TIA-1 RRM2,3-H94A H96A-Rev: AAAGACagcGAAagcATTGGGAATTCCTGGGGATCCACGCGG

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West D.L., Loughlin F.E., Rivero-Rodríguez F., Vankadari N., Velázquez-Cruz A., Corrales-Guerrero L., Díaz-Moreno I, & Wilce J.A. (2022). Regulation of TIA-1 Condensates: Zn2+ and RGG Motifs Promote Nucleic Acid Driven LLPS and Inhibit Irreversible Aggregation. Frontiers in Molecular Biosciences, 9, 960806.

Publication 2022

pGEX-4T-2

Cleavage Inverse pcr Mutations Oligonucleotide Plasmid Rrm2 Site directed mutagenesis Tct 1 Thrombin Tia 1

Corresponding Organization :

Other organizations : Australian Regenerative Medicine Institute, Instituto de Investigaciones Químicas

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Variable analysis

independent variables

Mutations (H94A, H96A, H94A H96A) in TIA-1 RRM2,3

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

controls

Positive control: Not mentioned
Negative control: Not mentioned

Annotations

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