Date of Award

1997

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Ronald J. Siebeling

Abstract

Hepatitis A virus (HAV), the causative agent of food and water borne acute hepatitis worldwide, is very difficult and expensive to propagate in tissue culture. Large scale production of HAV proteins in E. coli by recombinant DNA techniques would be invaluable for antisera production, biochemical analyses and in developing sensitive diagnostic tests. Therefore, PCR amplified DNA fragments encoding the HAV capsid proteins were cloned individually into prokaryotic expression vectors for production of either recombinant fusion or non-fusion proteins. Cloning and expression of HAV capsid genes, which induced toxic effects in certain host E. coli, was tolerated better by protease-deficient host cells. HAV capsid proteins expressed from the prokaryotic vector, pAX4b+, as $\beta$-galactosidase fusions reacted with antisera generated against intact HAV particles. However, fusion protein expression and subsequent affinity-based purification were complicated by co-expression of $\beta$-galactosidase and its cross-reactivity with anti-HAV antibodies. An attempt was made to improve transcription by incorporating the E. coli rrnB BoxA sequence in frame between the lacZ and HAV VP1 gene segments in the fusion construct. Induction of protein expression from recombinant pAX-BoxAVP1 did not reveal any HAV related protein but resulted in an overexpression of a 33 kD protein with a strong homology to protein sequences derived from the origin of replication of F and similar E. coli plasmids. HAV capsid sequences were cloned successfully into the pT7-7 vector for expression of proteins in a non-fusion form, and into the $\lambda$SurfZAP$\sp{\rm TM}$ vector for expression of a membrane-directed, surface-displayed fusion protein in E. coli and for the generation of chimeric coliphages. However, expression of a $\sp{\sim}$30kD protein following induction was seen in recombinants carrying pT7-VP3 plasmids. Expression of recombinant VP1 was not discernible in cells carrying either pT7-VP1 or pSurf-VP1. A study of the codon usage profiles revealed that the coding strategy in HAV might involve a higher frequency of codons recognized as rare in both E. coli and human systems, and may be responsible for the inefficient expression of HAV proteins in both E. coli and tissue culture.

ISBN

9780591614510

Pages

180

DOI

10.31390/gradschool_disstheses.6537

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