All abbreviations used in this study are listed in back matter. The cytokine TRAIL (TNF-related apoptosis-inducing ligand) from the TNF family is a promising anticancer drug that induces the apoptosis of cancer cells while leaving normal cells intact. In addition, animal cell culture media are about 100–1000 times more expensive than bacterial or fungal culture media. This indicates that the microbial systems are convenient and powerful tools for the production of recombinant proteins, despite the various bottlenecks and obstacles that they pose for the efficient production of functional mammalian proteins, namely the absence or unconventional post-translational modifications, proteolytic instability, poor solubility, etc. Most pharmaceuticals based on recombinant proteins are produced in microbial host cells such as bacteria or yeast.

SRH–DR5-B can be considered as a promising candidate for therapeutic applications. The resulting SRH–DR5-B fusion bound the VEGFR2 and DR5 receptors with high affinity and showed increased cytotoxic activity in 3D multicellular tumor spheroids. The replacement of His by Ser at position P2 immediately after the initiator Met dramatically minimized degradation, allowing more than 20 mg of protein to be obtained from 200 mL of cell culture. However, the HRH–DR5-B degraded during purification to form two adjacent protein bands on the SDS-PAGE gel. coli-optimized codons with AT-rich codons in the DNA sequence encoding the first 7 amino acid residues of the HRH peptide. Initially low expression of HRH–DR5-B was enhanced by the substitution of E. To enhance the efficacy of TRAIL, we designed the HRH–DR5-B fusion protein based on the DR5-selective mutant variant of TRAIL fused to the anti-angiogenic synthetic peptide HRHTKQRHTALH.

The recombinant anti-cancer cytokine TRAIL has shown a limited therapeutic effect in clinical trials. Unlike conventional small molecule or monotargeted drugs, bifunctional proteins have increased biological activity while maintaining low systemic toxicity. In the last two decades, bifunctional proteins have been created by genetic and protein engineering methods to increase therapeutic effects in various diseases, including cancer.

Graphpad prism 8 trial