IL-2 cytokine therapy is made safer by splitting a designed mimetic in two for tumor targeting.
Synthetic gene construction The designed protein sequences were codon optimized for Escherichia coli expression and ordered as synthetic genes in pET29b + E. coli expression vectors (Genscript). Each synthetic gene was inserted at the NdeI and XhoI sites of the vector, including an N-terminal hexahistidine tag followed by a TEV protease cleavage site and the addition of a stop codon at the C terminus. VHH fusion proteins were inserted in pCMV/R for mammalian cell expression cloned between NheI and AvrII sites and including an N-terminal signal peptide (MDSKGSSQKGSRLLLLLVVSNLLLPQGVLAGSDG) (Genscript). Amino acid sequences are listed in Supplementary Tables 1–3. Protein expression and purification in E. coli All DARPin protein fusions H132' and H32'4 were expressed in E. coli strain Lemo21(DE3) (NEB) and VHH fusion proteins in E. coli strain SHuffle T7 (NEB). Bacteria were transformed with a pET29b+ plasmid encoding the synthesized gene of interest. Cells were grown for 24 h in lysogeny broth medium supplemented with kanamycin. Cells were inoculated at a ratio of 1:50 ml in Studier TBM-5052 autoinduction medium supplemented with carbenicillin or kanamycin, grown at 37 °C for 2–4 h and then grown at 18 °C for an additional 18 h. Cells were harvested by centrifugation at 4,000g and 4 °C for 15 min and resuspended in 30 ml of lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.02 mg ml–1 DNAse). Cell resuspensions were lysed by sonication for 2.5 min (5-s cycles). Lysates were clarified by centrifugation at 24,000g at 4 °C for 20 min and passed through 2 ml of Ni-NTA nickel resin (Qiagen, no. 30250) pre-equilibrated with wash buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 30 mM imidazole). The resin was washed twice with ten column volumes (CV) of wash buffer and then eluted with 3 CV of elution buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 300 mM imidazole). Eluted proteins were concentrated using Ultra-15 Centrifugal Filter Units (Amicon)…
Quijano-Rubio, Department Of Biochemistry, Institute For Protein Design, University Of Washington, Seattle, Department Of Bioengineering, Monod Bio, Inc., Bhuiyan, Aladdin M.