Document Type

Thesis

Date of Degree

Fall 2014

Degree Name

MS (Master of Science)

Degree In

Chemical and Biochemical Engineering

First Advisor

David W. Murhammer

Abstract

The baculovirus expression vector system (BEVS) is extensively used to produce recombinant proteins due to its high rate of expression. The major drawback of using this system is the early cell death (typically after 48-72 h post infection) that leads to decreased recombinant protein expression. Viral infection increases the production of reactive oxygen species (ROS) that are believed to contribute to this early cell death. Baculoviruses contain a Copper-Zinc Superoxide Dismutate (CuZnSOD) homolog gene that inactivates cellular CuZnSOD activity in insect cells by ~48 h post infection. Specifically, the CuZnSOD homolog inactivates the CuZnSOD enzyme by binding the copper chaperone, thereby leading to increased oxidative stress and presumably more rapid cell death. CuZnSOD activity during Wt-AcMNPV infection decreased to 0 in about 48-60 h post infection.

In this study the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) modified to overexpress human CuZnSOD and Copper chaperone cassette (CCS) and devoid of the viral CuZnSOD homolog showed lower infectivity compared to Wt- AcMNPV infection. Futhermore, the addition of H2O2 to induce oxidative stress increased the infectivity of the modified AcMNPV, thereby supporting the premise that a minimal level of oxidative stress is required for improved infection. Further investigations are required to determine if this modified virus would be a better expression vector then the conventionally used baculovirus vectors.

Public Abstract

Baculoviruses are widely used with insect cells to express recombinant proteins. While the rate of recombinant protein expression can be very high in this system, the protein expression ceases within 2-3 days due to cell death resulting from the virus infection. Evidence suggests that oxidative stress is a major contributor to this cell death.

A baculovirus-encoded Copper-Zinc Superoxide Dismutate (CuZnSOD) homolog gene contributes to this oxidative stress. Specifically, the CuZnS OD homolog inactivates the CuZnSOD enzyme by binding to copper chaperone, thereby leading to increased oxidative stress and presumably more rapid cell death. Cell longevity potentially can be extended by increasing cellular antioxidant defenses, e.g., by removing the CuZnSOD homolog and over expressing active CuZnSOD. The current research investigates the role of this homolog by studying modified baculovirus in which the CuZnSOD homolog gene has been removed and Human CuZnSOD is overexpressed.

Keywords

publicabstract

Pages

xii, 56 pages

Bibliography

Includes bibliographical references (pages 54-56).

Copyright

Copyright 2014 Bhakti Kishor Bapat

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