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PRODUCTION AND CHARACTERIZATION OF PROINSULIN-TRANSFERRIN FUSION PROTEIN
by
Yu-Sheng Chen
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
August 2011
Copyright 2011 Yu-Sheng Chen
Object Description
| Title | Production and characterization of proinsulin-transferrin fusion protein |
| Author | Chen, Yu-Sheng |
| Author email | yushengc@usc.edu;cys840227@gmail.com |
| Degree | Master of Science |
| Document type | Thesis |
| Degree program | Pharmaceutical Sciences |
| School | School of Pharmacy |
| Date defended/completed | 2011-06-22 |
| Date submitted | 2011-07-16 |
| Date approved | 2011-07-18 |
| Restricted until | 2011-07-18 |
| Date published | 2011-07-18 |
| Advisor (committee chair) | Shen, Wei-Chiang |
| Advisor (committee member) |
Okamoto, Curtis Toshio Wang, Clay C. C. |
| Abstract | A transferrin-based fusion protein, proinsulin-transferrin (ProIns-Tf) had been constructed using recombinant protein technology in our laboratory. Based on the in vitro preliminary results, ProIns-Tf reduced glucose production in H4IIE liver cells and increased glucose uptake in adipocytes. These potent effects were achieved by conversion of the ProIns moiety to an active Ins-like moiety through the mechanism of TfR-mediated endocytosis. Therefore, ProIns-Tf showed great potential for diabetes therapeutics. However, it was also observed that the preparation of this fusion protein also produced a large amount of protein impurities which might have impeding effects on the further characterization studies. ❧ In this report, ProIns-Tf fusion protein was purified using the polyhistidine tag technique. The hexahistidine tag (6xHis tag) was bound to the C-terminus of Tf domain through mutagenic polymerase chain reaction (PCR) and recombinant DNA technology. After the protein expression and purification processes, the protein analysis results indicated that the purity of ProIns-Tf was significantly enhanced. In addition, through the transferrin receptor (TfR) competitive binding assay, we found that the insertion of C-terminal 6xHis tag did not interfere with the binding affinity of Tf domain to TfR. Therefore, it was concluded that the His-tag purification is a suitable approach to improve the purity of Tf-based fusion protein. ❧ Furthermore, by comparing the results to previous studies in our laboratory, we also noticed that the TfR binding affinity of our Tf-based fusion proteins - ProIns-Tf, granulocyte colony-stimulating factor- Tf (G-CSF-Tf) and human growth hormone-Tf (hGH-Tf) - had a positive correlation to the molecular weight of the bound protein domain. It also implies that the beneficial effects of the TfR-mediated endocytosis should be limited by the size of Tf-bound protein drug. As a result, molecular weight of protein drug will be considered as an important criterion for the design of future Tf-based recombinant fusion proteins. |
| Keyword | bifunctional fusion protein; his-tag; insulin; proinsulin; protein purification; transferrin |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m |
| Rights | Chen, Yu-Sheng |
| Access conditions | The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given. |
| Repository name | University of Southern California Digital Library |
| Repository address | USC Digital Library, University of Southern California, University Park Campus MC 7002, 106 University Village, Los Angeles, California 90089-7002, USA |
| Repository email | cisadmin@usc.edu |
| Archival file | uscthesesreloadpub_Volume71/etd-ChenYuShen-99.pdf |
Description
| Title | Page 1 |
| Full text | PRODUCTION AND CHARACTERIZATION OF PROINSULIN-TRANSFERRIN FUSION PROTEIN by Yu-Sheng Chen A Thesis Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE (PHARMACEUTICAL SCIENCES) August 2011 Copyright 2011 Yu-Sheng Chen |
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