Please use this identifier to cite or link to this item: http://prr.hec.gov.pk/jspui/handle/123456789/15583
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dc.contributor.authorShahzadi, Syeda Kiran-
dc.date.accessioned2020-11-11T07:48:48Z-
dc.date.available2020-11-11T07:48:48Z-
dc.date.issued2020-
dc.identifier.govdoc20719-
dc.identifier.urihttp://prr.hec.gov.pk/jspui/handle/123456789/15583-
dc.description.abstractCancer is first cause of death in developed countries and the second cause of death in developing countries. It was reported that approximately 12.7 million cancer cases occur worldwide with 7.6 million of mortality rate. Human IFN-α2b is approved by the FDA as a therapeutic protein that can be used to treat some types of cancer. Development and further exploration of human IFN-𝛼2b to improve its efficacy and safety is very important as it may lead to the improvement of patient’s life quality In the present research work, attempts were made to produce biological active modified forms of human IFN-α2b and PEGylate them to increase their in vivo circulation half-life. Initially, human IFN-α2b gene was doubly cut from pTZ-IFN (gifted) and was ligated into pET 21a vector for expression in E. coli BL21 Codon Plus competent cells. Later on, modified forms of IFN-α2b (IFN-1, IFN-2, IFN-3 and IFN-4) were developed by sitedirected mutagenesis of human IFN-α2b gene at amino acids positions R22, R23 and H34. The wild type and modified forms of human IFN-α2b were expressed in E. coli, refolded, purified by Q-sepharose resin and characterized by SDS-PAGE, HPLC and MALDI-TOF. Human IFN-α2b and its modified forms were PEGylated with 20 and 40 kDa mPEGPropionaldehyde and mPEG-Succinimidyl Succinate. PEGylated products were then purified using SP-sepharose and Fractogel EMD CM 650(M) resin respectively and characterized by SDS-PAGE, HPLC and MALDI-TOF. Antiproliferative activities of all products were studied using HepG2 cell line and circulation half-life was determined in vivo in Rabbits. Molecular docking studies with IFNAR1, IFNAR2 (receptors) and PEGs vii was also performed to determine the effect of mutations and PEG molecules on binding of IFN-α2b with its receptors. Experimentally produced IFN-α2b inhibited HepG2 cancerous cells growth at concentration 0.125 ng and was comparable to Refron which achieved IC50 at 0.125 ng. IFN-2 (R(23)H) achieved IC50 at 0.062 ng and thus slightly greater biological activity than normal IFN-α2b. However, R(22)H modification in IFN-4 resulted in production of modified form having IC50 at 0.125 ng that was comparable to normal IFN-α2b. In case of IFN-1, modification at R(23)A and H(34)R, biological activity was decreased significantly (IC50: 1 ng). Similarly, modifications at R(22)A and H(34)R) in case of IFN3 also decreased the biological activity (IC50: 0.5 ng), which was higher than normal IFNα2b but not to the extent as IFN-1. Experimental findings showed that PEGylation of IFNα2b and its modified forms with linear and branched PEGs (20 kDa and 40 kDa respectively) has increased their hydrodynamic volume and also increased the serum retention time (up to 62 hours) as compare to normal wild type IFN-α2b (4 hours). However, antiproliferative activity in HepG2 cell line was decreased (49 % to 7 % as compare to IFN-α2b wild type (100%)) with increase in PEGylated IFN-α2b size. Molecular docking studies revealed that increase in PEGylated IFN-α2b size (from 19 kDa to 60 kDa), the receptor binding site on IFN-α2b was sterically shielded resulting in low biological activity of PEGylated IFN-α2b. These results depicted the significance of amino acid position number 22, 23 and 34 in determining the biological activity of IFN-α2b. PEGylation resulted in protein species with reduced biological activities. However, in vivo circulation half-life of all PEGylated interferons was increased up to many folds due to increase in molecular weight after PEGylation.en_US
dc.description.sponsorshipHigher Education Commission Pakistanen_US
dc.language.isoenen_US
dc.publisherUniversity of the Punjab , Lahoreen_US
dc.subjectPhysical Sciencesen_US
dc.subjectChemistryen_US
dc.titleModification and Pegylation of Human Interferon alpha-2b for Enhanced Biological Activityen_US
dc.typeThesisen_US
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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