Figure 1.

Construction of a vector insert for expression of a single chain IL-12 fusion protein. (A) Schematic of the DNA construct encoding for a single chain IL-12 fusion protein. The p35 and p40 subunits were genetically fused with a DNA linker encoding for the 15 amino acids (glycine₄ serine)₃ using the SacI and EcoRV restriction sites. (B) To assure secretion in eukaryotic cells, an OKT3 leader sequence was introduced upstream from the p35 gene. For this purpose, the BalI and SmaI restriction sites were used, replacing the N-terminal arginine of p35 with two glycine residues downstream from the cleavage site, as indicated by the black arrow. (C) Fusion of p35 and p40 with a synthetic DNA linker was established by use of a naturally occurring SacI site at the 3′-end of the p35 gene and an engineered EcoRV site upstream from the 5′-end of the p40 gene, which introduced additional aspartic acid and isoleucine residues.