Doctor of Philosophy, The Ohio State University, 2023, Chemical Engineering

Since its discovery in the late 20th century, inteins have been extensively explored and utilized in various fields of biotechnology. Among the different types of inteins found in nature, the continuous intein is the most prevalent and exhibits splicing activity immediately upon transcription. Engineered cleaving continuous inteins can be utilized for coupling with various tags to develop self-cleaving tags to purify recombinant proteins. Additionally, they can be applied to novel approaches in drug delivery. However, the uncontrolled cleaving activity during expression poses a challenge by causing premature cleavage, thereby impeding their development and application to some extent. In this study, the extein dependence of the ∆I-CM intein was characterized using the on-column cleaving kinetic test. Guidelines were provided for predicting or modulating the cleavage rate of the ∆I-CM intein based on the extein sequence. A comparison of extein dependencies between the ∆I-CM intein and other commercially available C-terminal cleaving inteins was conducted, highlighting the similarities and differences among different inteins. Furthermore, insights into the mechanism of the extein affect C-terminal cleavage were provided. In this study, comprehensive characterization of two novel mutants of the Mtu RecA ∆I-CM intein obtained through yeast surface display for improved protein purification was performed using the on-column cleaving kinetic test. The two mutants, ∆I-12 and ∆I-29, exhibited significantly reduced temperature sensitivity while retaining pH sensitivity. Self-cleaving tags developed by combining these mutants with CBD tags and ELP tags can be applied for simple chromatography or non-chromatography purification of recombinant proteins. When applied in an E. coli expression system, these mutants showed a significant reduction in premature cleavage compared to the original Mtu RecA ∆I-CM intein. Furthermore, the successful development and deployment of a (open full item for complete abstract)

Committee: David Wood (Advisor); Shang-Tian Yang (Committee Member); Jeffrey Chalmers (Committee Member) Subjects: Chemical Engineering

Master of Science, The Ohio State University, 2021, Chemical Engineering

Bridging the gap between protein purification platforms at the laboratory scale and at the industrial scale remains a challenge, as there is no “one size fits all” technology that works for every protein. To date, only technology based on Protein A has managed to enter mainstream usage both in the lab and in industry, but it only works for monoclonal antibody and Fc fusions. Affinity chromatography can be used to purify most proteins, but a major downside of this is that they require the use of affinity tags – proteins purified with these tags must undergo further treatment to remove the tags, which is impractical at the industrial scale. Research into intein-based chromatography offers a possibility to get around this: engineered inteins can be used as a “self-cleaving” affinity tag that leaves a tagless target protein after elution. However, the rate at which inteins cleave themselves off a protein varies wildly depending on the target protein. This work aims to reduce that dependency by introducing a mutation known to reduce the intein's dependence on the +2 C-extein residue in the natural splicing version of the Npu intein into the engineered cleaving version to determine if it can also reduce its dependency on the +2 residue. The experiments performed in this work did not detect a significant difference in variance, however. In addition, it also appeared to slow down the overall cleavage kinetics, suggesting that the intein cleavage reaction is subject to different controls than the splicing reaction.

Committee: Andre Palmer (Committee Member); David Wood (Advisor) Subjects: Chemical Engineering