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Design and Production of a Fusion Pr...

Kates, Sydney.

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Design and Production of a Fusion Protein for the Treatment of Osteoarthritis.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Design and Production of a Fusion Protein for the Treatment of Osteoarthritis./
Author:	Kates, Sydney.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2019,
Description:	69 p.
Notes:	Source: Masters Abstracts International, Volume: 80-12.
Contained By:	Masters Abstracts International80-12.
Subject:	Biomedical engineering. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=13865167
ISBN:	9781392223697

Design and Production of a Fusion Protein for the Treatment of Osteoarthritis.
Kates, Sydney.

Design and Production of a Fusion Protein for the Treatment of Osteoarthritis. - Ann Arbor : ProQuest Dissertations & Theses, 2019 - 69 p.

Source: Masters Abstracts International, Volume: 80-12.

Thesis (M.S.)--Northeastern University, 2019.

This item must not be added to any third party search indexes.

Osteoarthritis (OA) is a degenerative disease of the whole joint, affecting millions worldwide. Over time, the cartilage, synovium, and bone can degrade, leading to severe inflammation and pain. Treatments for OA are for remediating pain and are not effective at a long-term scale. To create a more effective treatment, it is necessary to improve drug delivery. The way we hope to do this is by using charge interactions with cartilage and a drug carrier to create a long lasting, drug depot within the highly negatively charged cartilage.For the drug carrier, we use Avidin (net charge +20), and for therapeutics, we use Interleuin-1 Receptor Antagonist (IL-1RA) or Insulin-like Growth Factor 1 (IGF-1). Avidin has been proven as a good carrier for OA drugs as there is a high retention time in cartilage. Both IL-1RA and IGF-1 have been proven to reduce or halt the progression of OA.To create a therapeutic that is bound to a drug carrier, we used well-established bacterial expression systems. We designed an insert to produce our genes of interest with the incorporation of additional elements to allow for efficient use of the bacterial vector. We incorporated restriction enzyme sites that allow for replacement of the therapeutic gene of interest to allow for production of another desired compound. Restriction sites are also present to allow for creation of controls for these therapeutics where they are not bound to the Avidin carrier. A His tag was incorporated for purification steps, and a flexible linker was chosen to allow for activity at the binding sites of protein.We incorporated the DNA of interest into different bacterial vectors and E.coli cell lines to create our protein. For cloning, we used the pUC57-Kan vector and DH5α cells. For protein production, we used the pET11 vector and BL21 cells.We were able to produce what we believe to be a monomeric Avidin bound to IL-1RA, and the IL-1RA control. Due to difficulty of assaying the protein, we were not able to confirm that Avidin had formed its native tetrameric structure. Further assaying and in vitro testing will assess the protein efficacy.

ISBN: 9781392223697Subjects--Topical Terms:

535387
Biomedical engineering.

Design and Production of a Fusion Protein for the Treatment of Osteoarthritis.
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245 1 0 $a Design and Production of a Fusion Protein for the Treatment of Osteoarthritis.
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300 $a 69 p.
500 $a Source: Masters Abstracts International, Volume: 80-12.
500 $a Publisher info.: Dissertation/Thesis.
500 $a Advisor: Bajpayee, Ambika G.
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506 $a This item must not be added to any third party search indexes.
506 $a This item must not be sold to any third party vendors.
520 $a Osteoarthritis (OA) is a degenerative disease of the whole joint, affecting millions worldwide. Over time, the cartilage, synovium, and bone can degrade, leading to severe inflammation and pain. Treatments for OA are for remediating pain and are not effective at a long-term scale. To create a more effective treatment, it is necessary to improve drug delivery. The way we hope to do this is by using charge interactions with cartilage and a drug carrier to create a long lasting, drug depot within the highly negatively charged cartilage.For the drug carrier, we use Avidin (net charge +20), and for therapeutics, we use Interleuin-1 Receptor Antagonist (IL-1RA) or Insulin-like Growth Factor 1 (IGF-1). Avidin has been proven as a good carrier for OA drugs as there is a high retention time in cartilage. Both IL-1RA and IGF-1 have been proven to reduce or halt the progression of OA.To create a therapeutic that is bound to a drug carrier, we used well-established bacterial expression systems. We designed an insert to produce our genes of interest with the incorporation of additional elements to allow for efficient use of the bacterial vector. We incorporated restriction enzyme sites that allow for replacement of the therapeutic gene of interest to allow for production of another desired compound. Restriction sites are also present to allow for creation of controls for these therapeutics where they are not bound to the Avidin carrier. A His tag was incorporated for purification steps, and a flexible linker was chosen to allow for activity at the binding sites of protein.We incorporated the DNA of interest into different bacterial vectors and E.coli cell lines to create our protein. For cloning, we used the pUC57-Kan vector and DH5α cells. For protein production, we used the pET11 vector and BL21 cells.We were able to produce what we believe to be a monomeric Avidin bound to IL-1RA, and the IL-1RA control. Due to difficulty of assaying the protein, we were not able to confirm that Avidin had formed its native tetrameric structure. Further assaying and in vitro testing will assess the protein efficacy.
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