Structural and functional characterization of human serum albumin engineered variants produced in E. coli

Human serum albumin (hSA) is the most abundant protein in human plasma, and it represents the main carrier for endogenous and exogenous ligands including drugs and lipids. Thanks to the interaction with the human neonatal Fc receptor (hFcRn), hSA has a long serum half-life, which is up to three weeks. Due to this property, the design of compounds capable of binding hSA or the design of hSA fusion protein has gained a lot of interest in pharmacology to increase the serum half-life of the drugs under study. Three different variants of hSA (i.e., hSA1, hSA2, hSA3) have been developed using PROSS algorithm by Prof. Sarel Fleishman’s group (Weizmann Institute of Science, Rehovot, Israel), bearing respectively 16, 25 and 73 mutations, to be hyper stable, hyper soluble and expressible in bacterial host with yield up to 100 mg/L. Characterizing the binding of these hSA variants with hFcRn is of high interest in determining if these hSAs can be used as a platform for albumin-based drug discovery projects. In the present work I present: the production of hSA1, hSA2 and hSA3 in E. coli T7 shuffle and of a hFcRn soluble construct in ExpiHEK293 cells; the determination of binding affinity of both the hSA variants and wild type (wt) one towards hFcRn, by using surface plasmon resonance (SPR) technique and the isolation of complexes by using analytical size exclusion chromatography (SEC).