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Abstract

Background: Hepatitis C has emerged in recent years as a common cause of liver disease. An estimated 200 million people are thought to be infected worldwide. Hepatitis C virus (HCV) infection is characterized by viral persistence and chronic liver disease in approximately 80% of cases. Chronic HCV infection is curable by either a combination of interferon and ribavirin, or PEGylated interferon. Both drugs had been shown to inhibit the replication of the hepatitis viruses. Objectives: The work presented has two major objectives: 1) To clone, overexpress the interferon alpha2a and to produce the PEGylated form using codon optimized synthetic interferon alpha 2a; 2) To produce a novel variant of interferon and PEGylated interferon with ultra-activity. Methods: A synthetic gene coding for human interferon alpha 2a was codon-optimized and synthesized for maximum expression in E. coli using the vector pET28a.The recombinant protein was expressed and purified in the single step by Ni²+ charged column chromatography. The PEGylated interferon alpha 2a was prepared using the 40K polyethylene glycol. The purified product was confirmed using the MS and Moldi. DNA shuffling was performed to produce a novel variant of interferon. Results: Our work indicates that the enzyme was expressed to ~60% of the total host protein and that purification of the recombinant His-tagged protein could be achieved in a single step. The synthetic recombinant human interferon alpha2a expressed within this system was biologically active. We successfully then managed to produce a biologically active PEGlated interferon using a 40K polyethylene glycol. In an attempt to produce a novel variant of PEGylated interferon we prepared libraries of DNA fragments of interferon alpha2a gene using the DNA shuffling techniques. These libraries will be screened for more active interferon. The novel characterized variants will then be PEGylated and molecular studies on the novel PEGylated interferon will be performed. Conclusion: Our work has not only shown that a biologically active PEGylated interferon could be produced from a codon optimized synthetic interferon but also demonstrated that one of the very expensive drugs could be produced locally with the saving of tens of millions of dollars every year.

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/content/papers/10.5339/qfarf.2012.BMP40
2012-10-01
2019-09-17
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http://instance.metastore.ingenta.com/content/papers/10.5339/qfarf.2012.BMP40
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