Fusion to Albumin as a Means to Slow the Clearance of Small Therapeutic Proteins Using the Pichia pastoris Expression System

Of the numerous strategies that have been tested to slow the clearance of injected protein drugs from the body and circulation (1), fusion to albumin offers several advantages. Albumin is the most abundant protein in mammalian plasma and one of the longest lived. It lacks posttranslational modifications, with the exception of extensive disulfide bonding (2). If albumin can be fused in-frame with a therapeutic protein as a single-chain polypeptide, the novel protein may acquire the slow clearance profile of albumin, while retaining the activity important for clinical use. This acquisition derives primarily from an increase in the molecular volume of the therapeutic protein, such that it is no longer subject to loss via the kidneys. This approach has the potential to provide a more consistent and less heterogeneous product than one obtained, for instance, by chemical modification with polyethylene glycol. Whereas others have used Kluveromyces (3,4) and Saccharomyces (5) yeast species to produce human serum albumin (HSA) fusion proteins, we have used the methylotropic yeast, Pichiapastoris, to produce rabbit serum albumin (RSA) fusion proteins. This system is particularly well suited for albumin production (6). This chapter summarizes our experience gained in expressing hirudin (7), barbourin (8), and reiterated RSA fusion proteins (9) in this system.