Enhanced expression and distinctive characterization of a long-acting FGF21 and its potential to alleviate nonalcoholic steatohepatitis

We have previously constructed a novel polypeptide, PsTag, that should be useful in the development of biologics with properties comparable to those achievable by PEGylation, but with potentially less side effects. However, the low fermentation yields of polypeptide fusion proteins may limit the application of this technology. We suspected that when polypeptide fusion protein was expressed in E. coli, the corresponding 8 tRNAs were needed to transport a large number of repetitive 5 amino acids to the ribosomes and thus, resulting in a relative deficiency of these tRNAs. PsTag600-FGF21, a long-acting FGF21 fusion protein, was used as a model for studying the effects of these non-rare tRNAs on the efficiency of heterologous protein production in E. coli. To further enhance the expression level and facilitate purification, secretory expressions of PsTag600-FGF21 were achieved by fusion with three signal peptides. Meanwhile, a comparison of several distinctive characterizations was carried out between PsTag600-FGF21 and PEG20K-FGF21. We investigated the protective effects of PsTag600-FGF21 in a nonalcoholic steatohepatitis model induced by methionine- and choline-deficient diet. Our results showed that the provision of 8 tRNAs and secretory expression remarkably increased the expression levels of PsTag fusion protein, meanwhile there were no significant effects on E. coli growth states. PsTag600-FGF21 had a larger hydrodynamic volume, a higher affinity and a longer plasma half-life than PEG20K-FGF21, while avoiding vacuole formation in mice. In NASH mice, administration of PsTag600-FGF21 reduced hepatic steatosis, fibrosis and inflammation. Therefore, PsTag600-FGF21 with higher expression level may be further developed for potentially application in clinics. •A non-rare tRNAs supplementation system for protein production is proposed.•The signal peptide CGTase improves the expression level of PsTag600-FGF21.•PsTag600-FGF21 has a longer plasma half-life than PEG20K-FGF21.•PsTag600-FGF21 does not produce detectable kidney vacuoles.•PsTag600-FGF21 reduced hepatic steatosis, fibrosis and inflammation.