*4.1. Cloning, Expression, and Purification of the (I27)4\_AS\_(I27)4 Polyprotein*

In order to obtain a (I27)4\_AS\_(I27)4 polyprotein, consisting of a single AS molecule, flanked by four repetitions of titin immunoglobulin-like domain (I27) at the N-terminus and at the C-terminus, the cDNA of the human *AS* (NP\_000336) was cloned in the pRSet.A(I27)8 expression vector [47], taking advantage of the NheI restriction site placed in the middle of (I27)8 encoding sequence. A mutagenic PCR was performed on the pEGFP\_AS vector [58] to delete the start and stop codons and to insert a NheI restriction site at both extremities of the *AS* gene. The PCR was carried out using the Q5®High-Fidelity DNA Polymerase (NEB, cat. #M0491) with the following primers: forward primer 5' AAAAGCTAGCGATGTATTCATGAAAGGAC 3', reverse primer 5' AATTGCTAGCGGCTTCAGGTTCGTAG 3', (in bold, the NheI restriction site). After sequencing, the pRSet.A (I27)4\_AS\_(I27)4 vector was used to transform BL21(DE3) *Escherichia coli* cells. Transformed cells were grown in Luria-Bertani medium at 37 ◦C until they reached an OD600 of 0.4–0.6 and the expression of the polyprotein was induced overnight at 22 ◦C by the addition of 1 mM IPTG. Cells were subsequently harvested by centrifugation and resuspended in lysis buffer (50 mM Na2HPO4, 300 mM NaCl, 10 mM imidazole, 4% Triton™ X-100, and 0.5 mM phenylmethylsulfonyl fluoride) before sonication on ice. The purification was performed by gravity flow column ion metal affinity

chromatography (IMAC), taking advantage of the 6× His-tag present at the N-terminus of the polyprotein. The soluble fraction of cell lysate was incubated on Ni+-NTA resin (Roche, cat. #05893682001) for 1 h at 4 ◦C with gentle agitation. The washing step was carried out in 50 mM Na2HPO4, 300 mM NaCl added with 20 mM imidazole, elution was achieved in the same buffer, added with 250 mM imidazole. The presence of the protein in the eluted fractions was verified by SDS-PAGE on a 4–12% polyacrylamide gel (InvitrogenTM, ThermoFisher Scientific, cat. #NW04120BOX) stained with Coomassie Brilliant Blue.
