Studies On Degradation Of HSA Fusion Protein Expressed In Pichia Pastoris

The technology of human serum albumin (HSA) fusion protein/peptide is currentlyconsidered one of the most potential applications to extend the drug half-life, and the Pichiapastoris expression system is preferred to express HSA fusion protein. However, preliminarystudies found that most all of the HSA fusion proteins degraded when expressed in P. pastoris.This not only reduces the yield and activity of the target protein but also brings moredifficulties for post-purification.The reasons of HSA fusion proteins degradation when expressed in P. pastoris werestudied in the paper with two fusion proteins IL2-HSA and HSA-IFNβ, which were expressedwell in our laboratory. Comparing the intracellular proteins extracted from the recombinant P.pastoris with those in broth indicated that the HSA fusion proteins had degraded inside thecell during expression. The degradation fragments of66kDa, IH-66and Hβ-66, which werecommon in these two HSA fusion proteins, were purified respectively from fermentativebroths. The results of Western blot, MALDI-TOF/TOF MS and N-terminal amino acidsequence analysis revealed that HSA fusion proteins were degraded near the connectionpoints between HSA and pharmacal proteins．Large amount of45kDa and61kDa fragments appeared respectively in the process ofHSA-IFNβ and IL2-HSA expression. Western blot analysis revealed that the former can behybridized by anti-HSA polyclonal antibody and almost not by anti-IFNβ polyclonal antibody,while the latter can be hybridized by anti-HSA and anti-IL2polyclonal antibodies.Considering the Immunogenicity and molecular weight of each fragment, they are generatedby the characteristic degradation of HSA. Therefore, the stability of HSA is important forHSA fusion proteins. Accordingly, site-directed mutagenesis (R410A, K413Q, and K414Q) ofHSA was carried in the research. The Mutant mHSA was expressed respectively by P. pastorisGS115and Kluyveromyces lactis GG799and the degradation has been improved. The causesand solutions to degradation of HSA fusion proteins require further study.