Cloning and protein purification

Prokaryotic expression constructs for different titin fragments and E3-ligases were produced for expression in E. coli and subsequent affinity purification. DNA fragments were obtained by PCR using human cDNA. PCR products were ligated into pGEX-4 T-1 expression vector. The generated constructs for titin fragments according to NCBI accession numbers nm_003319 (N2B isoform), nm_133378 (N2A isoform) and nm_001256850.1 (N2BA-Isoform) were as follows: N2-B region (exon 49), N2A (exons 102–109), a constitutively expressed PEVK segment (exons 219–225) and A168-170 (Ig141/Ig142/FN3-132). Human E3 ubiquitin ligases MuRF-1 (= TRIM63, nm_032588.4), MuRF-2 (= TRIM55, nm_184085.2), MuRF-3 (= TRIM54, nm_187841.3), CHIP (= Stub1, nm_005861.4) and Fbx32 (nm_148177) were cloned in pGEX-4 T-1 vector as well. All constructs were verified by Sanger-sequencing. Vectors were transformed in competent E. coli NEB Express (New England Biolabs) and expression was induced using 0,2 mM isopropyl-ß-d-thiogalactopyranoside. Fragments were purified using glutathione affinity chromatography. Thrombin (10–20 U) cleavage separated the titin fragments from their GST-tag. Para-aminobenzamidine sepharose beads were used to eliminate thrombin from the solution.