Simplified protease-free bioseparation methods by self-cleaving inteins.

摘要

Self-cleaving inteins have become an attractive tool for affinity tag removal in the past decade. By pairing the self-cleaving intein to a thermally responsive biopolymer termed elastin-like polypeptide (ELP), a protease-free, non-chromatographic bioseparation system was created, resulting in a rapid, inexpensive, and flexible bioseparation method. In this dissertation, recent advances in the intein and ELP-intein mediated protein purification systems will be discussed. First, the previously developed DeltaI-CM intein was re-engineered to be compatible with Invitrogen's TopoRTM and GatewayRTM cloning systems, making efficient ligation of PCR-amplified genes and parallel examination of promoters or affinity tags simpler and faster. Next, the self-cleaving intein system was applied to the E. coli secretion scheme, purifying several secreted proteins successfully, including two disulfide-bonded human antibody fragments. Then, by examination of the Hofmeister series, we greatly improved the previously developed ELP-intein method by a simple salt substitution, reducing the required salt concentration for ELP precipitation by 73%. Finally, results for the mammalian expression of ELP-intein-tagged proteins are presented and discussed, along with genetic optimization efforts performed to lower the GC content, reduce repetitiveness and improve the stability of the ELP gene. Overall, the advances made in this dissertation further strengthen both the intein and ELP-intein mediated bioseparation systems, generating robust and inexpensive purification methods that could have a potential impact on future downstream processing technology used by the biopharmaceutical and other industries.