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HUMAN GROWTH HORMONE – TRANSFERRIN RECOMBINANT FUSION
PROTEIN FOR ORAL DELIVERY
by
Nurmamet Amet
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)
August 2008
Copyright 2008 Nurmamet Amet
Object Description
| Title | Human growth hormone-transferrin recombinant fusion protein for oral delivery |
| Author | Amet, Nurmamet |
| Author email | amet@usc.edu; nurmamet@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Pharmaceutical Sciences |
| School | School of Pharmacy |
| Date defended/completed | 2008-05-23 |
| Date submitted | 2008 |
| Restricted until | Unrestricted |
| Date published | 2008-08-06 |
| Advisor (committee chair) | Shen, Wei-Chiang |
| Advisor (committee member) |
Haworth, Ian Kalra, Vijay |
| Abstract | Protein drug delivery is limited to needle injection associating with pain, inconvenience, and non- compliance. Therefore, protein drug with oral dosage form is preferred. We used transferrin (Tf) receptor mediated transcytosis and Tf-based recombinant fusion protein approach to achieve oral delivery of human growth hormone (hGH). Plasmid constructs expressing the fusion proteins were established by fusing coding sequences of both hGH and Tf in frame. Fusion proteins were produced in serum free media by transcient transfection of human embryonic kidney cells. The SDS-PAGE of conditioned media showed that fusion proteins expressed at ~ 90% abundance and 100 kDa molecular weight; the Western blot analysis with anti-hGH and anti-Tf antibodies verified the identity. The Nb2 cell proliferation and TfR binding assays demonstrated that fusion proteins retained bioactivity for both hGH and Tf, respectively. To minimize inter-domain interference, we inserted 2 copies of helical linker as a spacer between hGH- and Tf-domain, and discovered that it not only enhanced the bioactivity but also led to high-level expression (30-50 mg/L, in select fusion protein). Three fusion proteins (GT, GHT and THG) were assessed in hGH-deficient hypophysectomized rats for in vivo biological activity, with both subcutaneous and oral administration. 7-day s.c. administration in rats showed both GHT and THG fusion proteins promoted ~ 16 g body weight gain, whereas GT fusion protein led to ~ 12 g increase. However, when administered orally, only GHT fusion protein led to a small weight gain. We also investigated tryptic and chymotryptic stability of fusion proteins and found that these fusion proteins were highly susceptible to both enzymes, while Tf was resistant. 7-day oral coadministration of GHT fusion protein with soybean trypsin inhibitor resulted in modest body weight gain, with better consistency.; In conclusion, Tf-based recombinant fusion protein approach has a potential for oral delivery of protein drugs. |
| Keyword | fusion protein; oral delivery; helical linker; protein drug; human growth hormone; 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-m1548 |
| Rights | Amet, Nurmamet |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-Amet-2316 |
| Archival file | uscthesesreloadpub_Volume26/etd-Amet-2316.pdf |
Description
| Title | Page 1 |
| Full text | HUMAN GROWTH HORMONE – TRANSFERRIN RECOMBINANT FUSION PROTEIN FOR ORAL DELIVERY by Nurmamet Amet A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PHARMACEUTICAL SCIENCES) August 2008 Copyright 2008 Nurmamet Amet |
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