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Pham FINAL 12 7 2020 with cert.pdf (2.16 MB)

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Producing A Peptide For Use In A Blood Biosensor For Injury Detection

Pham, Errek Manh Trung
http://rave.ohiolink.edu/etdc/view?acc_num=ysu1607519672342672

Abstract Details

2020, Master of Science in Biological Sciences, Youngstown State University, Department of Biological Sciences and Chemistry.
Conventional hybridoma technology has been used for the selection and production of proteins for biosensor development. However, hybridoma technology is typically a slower and more costly procedure than phage display and produces a less durable end-product. Quicker and more efficient production of a small peptide using phage display has been studied at Youngstown State University for use in a blood biosensor. A heptapeptide BR-1 had been selected to bind to human serum albumin (HSA) with high specificity and was sequenced and inserted into a pMAL-c5X expression vector. In this study, biotinylation with biotin-PEG4-hydrazide and EDC chemistry, indirect enzyme-linked immunosorbent assays (ELISAs) with HSA target and biotinylated peptide (B-BR-1), and competitive inhibition peptide ELISAs with HSA target, soluble or bound Inhibition HSA, and B-BR-1 were used to confirm peptide specificity for HSA. The pMAL-c5X protein production and purification system, amylose resin affinity chromatography, 8–16% and 16.5% SDS-PAGE, and UV-Vis spectrophotometry were used to evaluate BR-1 production and purity. Binding of 2 µg B-BR-1 to 1 µg HSA target was significantly reduced by 0.01 and 0.1 µg bound Inhibition HSA (p < 0.05). Bound Inhibition HSA was found to inhibit B-BR-1 more strongly than soluble HSA. A fusion of maltose binding protein to BR-1 was produced and purified by affinity chromatography with a high yield of 2.23 mg/mL MBP/BR-1. However, evaluation of protein cleavage by SDS-PAGE and UV-Vis spectrophotometry was unable to detect soluble BR-1 peptide after cleavage. Optimization of BR-1 production and purification methods will enable future mass production of a small, durable peptide to develop a wearable blood biosensor to save the lives of military and law enforcement personnel.
Diana Fagan, PhD (Advisor)
Jonathan Caguiat, PhD (Committee Member)
David Asch, PhD (Committee Member)
150 p.
Biology; Chemical Engineering; Experiments; Immunology; Microbiology; Nanoscience; Nanotechnology
human serum albumin; biosensor; phage display; protein production; protein purification

Recommended Citations

Citations

  • Pham, E. M. T. (2020). Producing A Peptide For Use In A Blood Biosensor For Injury Detection [Master's thesis, Youngstown State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1607519672342672

    APA Style (7th edition)

  • Pham, Errek. Producing A Peptide For Use In A Blood Biosensor For Injury Detection. 2020. Youngstown State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ysu1607519672342672.

    MLA Style (8th edition)

  • Pham, Errek. "Producing A Peptide For Use In A Blood Biosensor For Injury Detection." Master's thesis, Youngstown State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1607519672342672

    Chicago Manual of Style (17th edition)

ysu1607519672342672
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This open access ETD is published by Youngstown State University and OhioLINK.