Recombinant Human CCL2 Production

The mature amino acid sequence for human CCL2 (aa 24–99) was obtained from NCBI, GeneID 6347. GBlocks (IDT) were created for this sequence with a C-terminal “LPETG” Sortase recognition site and complementary 5’ and 3’ overhangs to the NdeI/XhoI double digested pSTEPL Sortase fusion expression plasmid. ^{42 (link)} The resulting gBlocks were ligated into pSTEPL plasmids using Gibson assembly (NEB) and transformed into chemically competent SHuffle T7 Express E.coli (NEB). After bacterial liquid cultures were grown to 0.6 OD600, protein expression was induced overnight at 16°C with 0.2mM IPTG (Thermofisher, 15529019). The overnight cultures were lysed and sonicated in non-denaturing conditions: 20mM Tris, 125mM NaCl, 10mM imidazole, 0.1% Triton X-100 + COmplete tablet (Millipore Sigma, 11873580001), and the CCL2-LPETG STEPL fusion proteins were purified via Ni-NTA pulldown for PolyG-azidoester modification (Figures S1–2).