Date of Award

2019

Document Type

Thesis

Degree Name

Master of Science (M.S.)

Department

Biological Sciences

First Advisor

Geoff Lin-Cereghino

First Committee Member

Craig A. Vierra

Second Committee Member

Douglas C. Weiser

Abstract

Wounds in the mouth, occurring after oral surgery, take time to heal. No ointment can be added to help with the healing process because mouth saliva will constantly wash it away. In order to combat this problem, we propose engineering a normal flora microbe to grow at the site of injury and secrete a recombinant growth factor to promote healing of the damaged tissue. Our goal is to have the yeast Pichia pastoris produce human basic fibroblast growth factor (bFGF), which aids in cellular proliferation. P. pastoris is a good choice for this application because not only is it considered generally recognized as safe (GRAS) by the FDA, but it is a eukaryote that is able to perform posttranslational modifications and secrete large amounts of recombinant protein.

Previous studies have shown that a strain of P. pastoris can be engineered to express bFGF from a methanol-sensitive promoter. The study also showed that the bFGF, which was purified from the yeast’s extracellular medium, was able to promote the growth of NIH/3T3 cells (mice fibroblasts). Because we needed the P. pastoris to express the bFGF in glucose –based tissue culture medium in the presence of mammalian cells, we expressed the bFGF from the constitutive promoter GAP promoter. Along with optimizing and characterizing expression of bFGF, we also investigated the effect of the recombinant protein on mammalian cell growth using both scratch ad MTS assays. In addition, the effects of the yeast being co-cultured with mammalian cells was studied. Our results provide a basis for how a recombinant protein can be clinically used to improve wound healing in the mouth using a yeast strain to produce and secrete a growth factor at the site of injury.

Pages

73

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