Cloning and Expression of RNA polymerase L protein in Ebolavirus

Undergraduate #106
Discipline: Biological Sciences
Subcategory: Microbiology/Immunology/Virology

Natasha Hessami - Western Washington University
Co-Author(s): Sally Lyons-Abbott and Peter Myler, Center for Infectious Disease Research, Seattle, WA

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Ebolavirus is a Negative-Stranded RNA Virus (NSVs). EVD has seven protein domains, one of which is the very large L protein (polymerase): a multienzymatic polypeptide responsible for mRNA synthesis and modification, as well as the generation of progeny ribonucleoprotein complexes. By identifying the structure of the L-protein, it opens up the possibility of engineering a target-specific drug. We are cloning the L-protein gene for expression in yeast and mammalian cells after expression in E. coli failed. We have been cloning the L-protein of four different species (E. bundibugyo, Ebolavirus zaire, E. taï, and E. sudan) for expression, purification, and ultimately solution of the protein’s structure. The 4 genes of interest were amplified from a codon optimized vector for cloning into the yeast expression vector: pPICZB or pPICZBα. pPICZ vectors B and Bα are used for heterologous expression of proteins in Pichia pastoris. Gels were run with amplified inserts and empty vectors, and then we ran gels with the vectors after insertion had been attempted. By comparing the base length on the gel runs we were able to determine whether the insert had been taken up by the vector. Despite the larger size of the insert we were able to successfully amplify and clone three species into the pPICZ and pPICZα vectors. Successful cloned species and their respective vectors: E. bundibugyo – pPiczB and pPICZBα, E. sudan – pPICZBα, E. taï – pPICZB. Transform Pichia pastoris with sequenced construct and perform expression testing in Pichia pastoris. Once success has been reached in expression, purification scheme can be optimized and eventually a structure solution can be discerned through X – ray crystallography.

Funder Acknowledgement(s): SSGCID is supported by the National Institute of Allergy and Infectious Diseases [HHSN272201200025C and HHSN272200700057C]. Internship funded by University of Washington GenOM Project [NIH5R25Hg007153-03] Dr. Anne Dinning and Dr. Michael Wolf, and the Genentech Foundation.

Faculty Advisor: Lisa Peterson,