Protein Purification Techniques Using the Intein Self-Cleaving Model

College of Engineering Undergraduate Honors Thesis ScholarshipDenman Undergraduate Poster Forum: 3rd Place, 2015A central step in the production of high purity recombinant proteins is their separation and purification. Recombinant proteins are expressed in host cells from which they are collected and purified. Purification is necessary to separate target recombinant proteins from the unwanted contents of the host cells in which they are grown. Common methods employ several chromatographic steps, which requires optimization, the use of expensive resins, and large time investments. Another category of purification utilizes affinity tag sequences. Within this realm, affinity tags can be used in conjunction within the protein’s naturally occurring, self-cleaving intein. Using the intein simplifies the purification into a one-step chromatography purification. In this method, an affinity tag, characterized by its ability to selectively bind to a specified resin, is fused to a target protein. This allows for the protein to be selectively separated from unwanted proteins and cell debris. Then, a pH shift induces the intein’s self-cleaving capabilities, and the target protein can be separated from the resin and affinity tag. Non-chromatographic affinity tags exist which eliminate the need for affinity resins. The elastin-like polypeptide (ELP) tag is one such sequence. The use of ELP tag with the self-cleaving intein, makes it possible for purifications to be done independent of an affinity column. Shifts in salt concentration and pH lead to successful purifications of the target protein. ELP has a large and repetitive protein sequence which requires large amounts of energy for synthesis. By shortening the length of ELP, expression can be increased by freeing some of that energy. In order to test this, and to determine the optimal ELP tag length, which may be affected by size and solubility of the target protein, five different ELP tag sizes are studied. Another tag, the maltose binding protein (MBP) affinity tag, is commonly used in chromatographic purification processes due to its ability to selectively bind to immobilized amylose. The MBP-tagged target protein binds to an amylose resin. Then, a pH shift causes the intein to undergo a cleaving reaction, allowing the target tag to be separated from the affinity sequence. In an effort to increase the economic feasibility and simplicity of the MBP purification, the amylose resin is replaced with a starch solution. The backbone of starch is primarily composed of amylose units. Starch, which contains negatively charged ionic groups, can be easily salted out of solution. By allowing starch, in solution, to bind to an MBP-tagged target protein, the target protein can be separated from other proteins and cell debris via a salting out method. The target protein is further purified via a pH shift in the purification buffer. Because starch and salts are relatively cheap, the success of this approach will lead to a new, feasible option for mass purification and production of proteins. During the expression, proteins go through a folding process. In some cases, the recombinant proteins do not fold correctly, inactivating or altering the protein’s functionality. This is problematic in cases when the protein will be used to develop vaccines, such as the third protein discussed here. Initial results showed that, during column purification, the protein becomes aggregated so it cannot be recovered from the column’s affinity resin. In order to combat this problem, a Flag-Acidic-Target Tag (FATT) will be added to the protein using a polymerase chain reaction. FATT is made of three parts: the flag, the hyperacidic region and a target tag. The flag makes the tag easily detected. The hyperacidic region expresses well in E. coli, is also highly charged, so can be purified in a single step using a standard anion exchange chromatography resin, and most importantly, has been shown to promote correct protein folding during expression. It aids in proper refolding of misfolded fusion partners containing disulfide bonds due to the structure of the hyperacidic region, which acts as a shield-like non-specific chaperone for the target protein during in vitro expression and refolding. The tagged protein will be purified using column purification where it will selectively adhere to the column and be separated at a high purity. In all three experiments described here, protein yield will be determined using qualitative methods such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and quantitative methods, including activity and Bradford assays.The Ohio State University College of EngineeringNo embargoAcademic Major: Chemical Engineerin

Influences on Children\u27s Executive Function Skills Development

The development of executive function (EF) skills in young children is influenced by multiple factors; therefore, this paper focused on the association between home and school environmental influences. EF skills include working memory, inhibitory control, and attention or set-shifting cognitive abilities (Garon et al., 2008, as cited in Bernier, Carlson, Deschênes, & Matte-Gagné, 2012). It is important for parents and teachers to understand what EF skills are so these skills can be modeled for children. Parents and teachers also need to provide opportunities for children to practice these skills. With support from parents and other caregivers, children can learn to regulate emotions, behaviors, and cognitive states within the first three years, which leads to developmental changes from infancy and childhood to voluntary regulation of emotions and actions by the child (Williams & Berthelsen, 2017). Research was analyzed and synthesized for this paper providing an overview of literature on EF skills of young children, focusing on home and school environmental influences. Previous EF studies offered insight on home and school environments, positive and negative effects on EF development (academics and behavior), and suggested interventions. Understanding the relationship between developing EF skills, factors influencing them, and the three main EF components allows parents and teachers the opportunity to assist children in the development of EF skills. Research connecting regulatory competencies with academic achievement, social-emotional competence, and behavioral adjustment pointed out the importance of familial factors that can influence the development of self-regulation in young children. This knowledge can assist with prevention and intervention strategies for helping children with self-regulation (Zeytinoglu, Calkins, Swingler, & Leerkes, 2017)

World enough and time : ethnobotany and historical archaeology

This paper discusses the use of ethnobotany by historic archaeologists

Incorporating Engineering in High School FACS and Chemistry Class

This paper presents the preliminary development of engineering units for a year-long high school Food Science and Chemistry course. While the course is not intended as an engineering course, we explored ways that students could be introduced to the engineering design process, and other engineering concepts, as a way to motivate interdisciplinary thinking and inspire future exploration. As this project is in its early stages, we will present three potential units that incorporate engineering while meeting Next Generation Science Standards and Minnesota State Science Standards related to this course

The Archaeology of Fish Haul Creek, Hilton Head Island, Beaufort County, South Carolina: A Preliminary Statement and Recommendations

In May 1982 a potentially significant prehistoric archaeological site was identified on the northern end of Hilton Head Island by developer John Crago and his foreman, Jerre Wekhorst. This site, situated on the north edge of the Coggin and Fish Haul Creek marsh, was uncovered during the early stages of road construction in the Fish Haul subdivision (Baygall area of Hilton Head Island). Wekhorst collected abundant pottery as the work continued in the subdivision and took a small sample to the Charleston Museum, where we examined it for the first time in early June. This collection was notable not only for the mix of pottery (both Early and Middle Woodland period sherds were present), but also for the large size of the Early Woodland sherds

Chicora research contribution 6

In this paper, data from several recent studies conducted in the Charleston, South Carolina area are used to demonstrate that floral remains have a role to play in the reconstruction of foodways and, further, that both archaeology and history can contribute to the reconstruction. A discussion of the potential, and the problems, of ethnobotanical studies precede a discussion of the present data base

Charleston Museum archaeological contributions ; 27

Willtown was founded in the late 17th century on the banks of the South Edisto River, but the movement of the Willtown Church in the 1760s to another location marked the demise of the town. Hugh C. Lane Jr. encouraged The Charleston Museum in its research in and around the Willtown area, asking the question, "Why did Willtown fail?" "Our serendipitous discovery of James Stobo's rice plantation a mile from Willtown revealed a site remarkable in its pristine preservation, the clarity of its stratigraphic record, the number and types of artifacts recovered, and in the complexity of its architectural detail.

The Southern Colonial Backcountry: Interdisciplinary Perspectives on Frontier Communities

https://trace.tennessee.edu/utk_early-american/1014/thumbnail.jp

Cervicovaginal mucus barrier properties during pregnancy are impacted by the vaginal microbiome

IntroductionMucus in the female reproductive tract acts as a barrier that traps and eliminates pathogens and foreign particles via steric and adhesive interactions. During pregnancy, mucus protects the uterine environment from ascension of pathogens and bacteria from the vagina into the uterus, a potential contributor to intrauterine inflammation and preterm birth. As recent work has demonstrated the benefit of vaginal drug delivery in treating women’s health indications, we sought to define the barrier properties of human cervicovaginal mucus (CVM) during pregnancy to inform the design of vaginally delivered therapeutics during pregnancy.MethodsCVM samples were self-collected by pregnant participants over the course of pregnancy, and barrier properties were quantified using multiple particle tracking. 16S rRNA gene sequencing was performed to analyze the composition of the vaginal microbiome.ResultsParticipant demographics differed between term delivery and preterm delivery cohorts, with Black or African American participants being significantly more likely to delivery prematurely. We observed that vaginal microbiota is most predictive of CVM barrier properties and of timing of parturition. Lactobacillus crispatus dominated CVM samples showed increased barrier properties compared to polymicrobial CVM samples.DiscussionThis work informs our understanding of how infections occur during pregnancy, and directs the engineering of targeted drug treatments for indications during pregnancy