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

Doctor of Philosophy, The Ohio State University, 2018, Chemical Engineering

Recombinant therapeutic proteins changed the world over 30 years ago when insulin, the first therapeutic protein, was approved. Since then, over 200 therapeutic proteins have been approved to treat a wide range of diseases from diabetes to immune disorders. Currently, there is no universal platform that can be used to purify any given target protein in a quick and inexpensive manner. The self-cleaving split intein tag technology remedies this issue by creating a universal platform that can purify any traceless, tagless target protein rapidly and economically. Chapter 2 discusses the combination of the split intein purification strategy with cell-free protein synthesis (CFPS) systems to reduce the time it takes to produce therapeutic proteins. With the cell-free systems, proteins can be produced in hours compared to days or even weeks. The combination of CFPS and the split intein tag technology has been utilized in the creation of a device to produce biologics on demand. The BioMOD device aims to produce a single-dose of any therapeutic protein within 24 hours, specifically with a military application in mind. Chapter 3 discusses the use of magnetic beads to mediate the split intein purification. Combining the split intein and magnetic beads creates a more efficient purification process that requires less buffer and set-up time. Four target proteins are used to demonstrate the applicability of the system. Chapter 4 discusses the regeneration of a commercially available resin that has been used to covalently immobilize the N fragment of the split intein using a thioester bond. Due to the commercially available resin having a high price point and the lengthy amount of time it takes to immobilize the N fragment, regeneration of the resin was necessary. A panel of buffers was screened to find the best regeneration buffer. Using the best buffer, a life cycle analysis was done using 20 regeneration cycles to show the resin could be regenerated multiple times. The develo (open full item for complete abstract)

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

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

The biopharmaceutical market has seen tremendous growth over the past few decades as new recombinant proteins, monoclonal antibody-based therapeutics and gene therapies have been commercialized. After production in different mammalian, yeast and microbial expression hosts, each product must be concentrated and highly purified before clinical use. Protein A affinity methods provide a convenient and widely used platform for capturing and purifying monoclonal antibodies (mAbs), Fc-fusion proteins, antibody drug conjugates (ADCs) and bispecific antibodies (BsAbs). In this work, an attempt has been made to develop novel Protein A affinity ligands having higher binding capacity than the ligands on commercial resins. There is currently no similar platform technology for purifying increasingly important non-mAb protein therapeutics. Protein therapeutics such as single domain antibodies, single chain variable fragments, Fab fragments, interferons, epoetins, clotting factors, growth factors, insulin and insulin like analogues, enzymes etc. have traditionally been purified using multiple column steps based on ion exchange, hydrophobic interaction, mixed mode, and ceramic hydroxyapatite chromatography. These multicolumn approaches require significant optimization and often result in low product yields and recoveries. Thus, scalable and cost-effective alternatives to these currently used approaches are needed. Furthermore, these alternative methods should be convenient to use and allow for easy technology transfer between clinical drug discovery, process development and manufacturing. In this work, we propose the use of pH-sensitive self-removing affinity tags as a potential solution. Purification strategies based on self-removing and self-precipitating tags have been developed previously for laboratory scale protein purification. However, these methods utilize pH sensitive contiguous inteins which suffer from premature cleavage, resulting in significant product loss during p (open full item for complete abstract)

Committee: David Wood (Advisor); Eduardo Reategui (Committee Member); Jeffrey Chalmers (Committee Member) Subjects: Biochemistry; 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

Doctor of Philosophy, The Ohio State University, 2021, Chemical Engineering

Today, inteins have been extensively used in a wide variety of applications in biotechnology. Split inteins make up a small sector of inteins discovered more recently. Split inteins are expressed as two separate peptides naturally and are not active until intein-intein interaction. Due to their unique features, split inteins offer advanced controllability and flexibility in both self-cleaving and splicing activity. The natural split Npu intein was further engineered for pH controllability for applications including tag removal and on-column purification. The self-cleaving intein was first utilized as a tag removal platform for non-chromatographic aggregating tags. As a proof of concept, four target proteins were engineered with the aggregating beta-roll tag infusion with the pH controlled self-cleaving split intein. Beta-lactamase (beta-lac), super-folder green fluorescent protein (sfGFP), streptokinase (SK) and maltose binding protein (MBP), were successfully purified with the BRT17 intein fusion tag and produced tagless purified protein. The on-column purification strategy using the engineered self-cleaving split intein was employed for the production of valuable mammalian expressed target proteins and glycoprotein, ML39 single-chain variable fragments (scFv), Erythropoietin (EPO) and Interferon alpha-2b (IFN). All three protein were secreted, purified, and recovered at a high purity with a significant reduction of host-protein and DNA. Additionally, EPO and IFN glycosylation sites were further analyzed and verified. The use of nanotechnology in biomedical sciences has created a strong interest in its potential to dramatically improve the treatment and diagnosis of diseases. In collaboration with Columbus Nanoworks Inc, we developed simple and robust bioconjugation platforms for creating protein-labeled nanoparticles using split intein technology. In this work, we utilize two different split inteins, Gos-TerL and GP41.1, to achieve N-terminal and C-terminal or (open full item for complete abstract)

Committee: David Wood (Advisor); Jeffrey Chalmers (Committee Member); Eduardo Reátegui (Committee Member); Jennifer Ottesen (Committee Member) Subjects: Biology; Chemical Engineering; Molecular Biology; Pharmaceuticals

Doctor of Philosophy, The Ohio State University, 2018, Chemical Engineering

The development of platform technologies can dramatically impact the speed and ease with which new biological processes/products can be optimized and brought to market. Platform technologies offer researchers predictable, generalizable procedures for achieving a pre-defined outcome. This dissertation describes the development of two platforms for i) engineering RNA-based tools for gene regulation and ii) engineering self-cleaving intein affinity purification tags with more tightly-controlled cleavage kinetics for recombinant protein purification. Bacterial trans- acting small regulatory RNAs (sRNAs) have great potential for applications in the field of metabolic engineering, due to their modular nature and the relative ease with which they may be engineered for novel regulatory function at the level of mRNA translation control. Furthermore, these sRNAs act at their target mRNAs through relatively simple base-pairing interactions, and (in many cases) have been demonstrated to be portable from microbe to microbe, while also providing the benefit of tuning gene expression of multiple genes for optimization of metabolic pathway flux. Chapter 2 describes the development of a genetic system for engineering novel, multi-acting sRNA regulators derived from the E. coli-native DsrA sRNA. Chapter 3 establishes thorough design rules for these engineered sRNAs to ensure robust regulation of the target gene, and describes a design basis that provides these semi-synthetic sRNAs with unprecedented specificity for their target mRNAs. Chapter 4 describes the validation and use of a previously-established, yeast-surface display-based protein evolution platform for the engineering of self-cleaving intein affinity tags with applications in bioseparations. Affinity tag technology greatly simplifies the process of purifying diverse recombinantly-expressed proteins, but tag removal remains a non-trivial barrier to implementation of affinity capture for protein purification at scale. Affin (open full item for complete abstract)

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

Master of Science, The Ohio State University, 2016, Chemical Engineering

In biopharmaceutical industry, protein-based drugs have been rapidly growing over the past decade. Approximately 40%-90% of total cost of protein drug manufacturing is spent on downstream processing. Due to protein target complexity, multiple chromatography purification steps are required to obtain products with high purity. Dr. Wood's lab have been studying intein-mediated protein purification tag for years, one of the most significant discovery is the pH-sensitivity of intein cleaving reaction that result from zinc-binding motif. This work aims to investigate the mechanism of pH regulation on intein self-cleaving reaction and search for zinc-binding motif sequences with improved pH sensitivity.

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

Doctor of Philosophy, The Ohio State University, 2016, Chemical Engineering

Recent advances in upstream bioprocessing have largely increased the productivities of recombinant protein expression in various expression systems. This has led to an increasingly urgent need for rapid, cost-effective and large-scale downstream unit operations. Affinity tag-based purification provides an efficient platform to purify virtually any uncharacterized protein in a highly selective, high-throughput manner. With the combination of a self-cleaving intein, the purification and the tag removal processes can all be carried out in a single chromatography step, thus dramatically reducing the cost of downstream processing. Over the past decades, inteins have been genetically modified for better controllability and cleaving ability. However, unwanted in vivo premature intein cleaving remains a major bottleneck for applications in mammalian systems. In this dissertation, recent progresses in the control of intein cleaving will be discussed. A continuous delta I-CM intein and a zinc binding motif fusion allow the intein-tagged precursor proteins to be expressed in mammalian systems with less than 40-50% in vivo premature cleaving in the presence of 200 micro molar zinc chloride. An alternative, the Npu split intein system, has been shown to completely eliminate premature cleaving and has been altered into a pH sensitive cleaving intein. These efforts to control intein cleaving have led to the development of a robust and efficient platform technology for non-antibody recombinant protein downstream purification.

Committee: David Wood (Advisor); Andre Palmer (Committee Member); Shang-Tian Yang (Committee Member); Werner Tajrks (Committee Member) Subjects: Chemical Engineering

Doctor of Philosophy, The Ohio State University, 2016, Chemical Engineering

Since the 1980s, better understanding of protein structure and function has allowed for the design of useful proteins and their subsequent modification. In our laboratory, advances in protein engineering have been harnessed for the development of biosensors and the optimization of bioseparations. This dissertation encompasses work on both of these applications. First, previous work in our laboratory has developed a simple reporter protein in E. coli that can sense hormone-mimicking compounds of human nuclear hormone receptors (NHRs) and report their presence and activity through changes in growth phenotype. This system was extended from our current human targets to incorporate an insect NHR. The nuclear hormone receptor HR96 of both Aedes aegypti and Tribolium castaneum were designed, constructed, and optimized to validate novel bacterial biosensors for the detection of potential insecticides. This cell-based system was then incorporated into a cell-free expression environment to establish a faster colorimetric biosensing assay. Preliminary results show that our bacterial biosensor can be expressed in a cell-free protein synthesis reaction without compromising the sensitivity and selectivity of its response to known ligands. Second, our laboratory seeks to optimize the self-cleaving intein tag as a potential purification platform technology. We have previously shown that the combination of the elastin-like polypeptide (ELP) as a self-aggregating purification tag and the ΔI-CM intein can facilitate the purification of a target protein. This methodology not only eliminates the need of expensive chromatography resins but also eliminates the expenses associated with the use of proteases to recover the native protein after its purification. This promising tool was studied further to determine the optimal ELP tag length required to maximize both expression and purification yields. The shorter ELP tag lengths improved protein expression levels of three model target prot (open full item for complete abstract)

Committee: David Wood (Advisor); James Rathman (Committee Member); Jeffrey Chalmers (Committee Member) Subjects: Chemical Engineering

Doctor of Philosophy, The Ohio State University, 2015, Chemical and Biomolecular Engineering

The development of the Protein A platform for monoclonal antibody purification was one of the most important advances in biotechnology. However, no analogous system exists for the purification of non-antibody proteins. The use of self-cleaving intein tag technology is a potential platform for non-antibody protein purification. In this dissertation, recent advances in self-cleaving intein tag technology will be discussed. First, the ΔI-CM self-cleaving intein system was modified for use in high throughput protein purification applications. Second, the ΔI-CM intein was paired with a novel affinity tag, the choline-binding domain tag, to allow for affinity purification of recombinant proteins using commercially available anion exchange resins. This new purification was further optimized using a design of experiments approach. Finally, an ongoing project for the development of improved inteins will be presented and discussed. Currently, self-cleaving intein tag technology is limited to bacterial expression systems due to non-ideal intein cleaving kinetics. To be a useful platform, intein technology must be compatible with all types of expression hosts, especially mammalian cell culture. To overcome this, a method to engineer inteins with improved properties using directed evolution and yeast surface display was developed. This method was used to enrich libraries of intein variants for variants that are suitable for use in mammalian cell culture. Initial characterization of these enriched libraries identified several candidates with improved properties. Overall, the work described here lowers the barrier for widespread adoption of self-cleaving intein tag technology in the biotechnology and pharmaceutical industries.

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

Doctor of Philosophy, The Ohio State University, 2011, Biophysics

My research in The Ohio State University is focused on the development and application of structural and chemical biology approaches to probe protein function. In the first chapter, the challenges and advances in the field of membrane protein crystallography are discussed. X-ray crystallography is so far the most powerful technique to characterize the structural details of proteins. However its application on the membrane proteins was lagged due to the special properties of this class of proteins. New methodologies have been developed in recent years to facilitate membrane protein structure determination by adapting existing methods for soluble proteins. In the second chapter, the structure of an integral membrane protein, Rh protein, was determined by X-ray crystallography and its functional role and molecular mechanism were deduced and discussed from the structural data. Rh proteins, best known for their role as a human blood factor and antigen, are membrane channels that transport gas molecules, but it's in debating which, ammonia or CO2, is the physiological substrate of Rh protein. A structural biology approach is taken here to address this substrate issue by determining the first structure of an Rh family member, the Rh protein from Nitrosomonas europaea. This Rh protein exhibits a number of similarities to bacterial and archael ammonium transporters, including a trimeric state, a twin-His-centered central channel, and a Phe residue that blocks the channel. However, there are some significant differences, including an additional cytoplasmic C-terminal helix, an increased number of internal prolines along the transmembrane helices, as well as a specific set of residues linking the C-terminal helix to Phe blockage. This linkage suggests a possible mechanism in which binding of a partner protein to the C-terminus could regulate channel opening. Another difference observed in the Rh structure is the absence of the extracellular-cation binding site thought to recr (open full item for complete abstract)

Committee: Michael K. Chan PhD (Advisor); Ross E. Dalbey PhD (Committee Member); Charles E. Bell PhD (Committee Member) Subjects: Biochemistry; Biophysics