Publication Date

Fall 2019

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Engineering

Advisor

Claire . Komives

Keywords

Antivenom, Lethal Toxin Neutralizing Factor, LTNF, Pichia Pastoris, Rattlesnake, Tandem Repeats

Subject Areas

Chemical engineering; Biochemistry; Bioengineering

Abstract

It has been shown that a peptide of the first 10-15 N-terminal amino acids of the

lethal toxin neutralizing factor (LTNF) protein found in opossums (Didelphis spp.) holds

promise as a low-cost therapy for snake envenomation. To date, the 11-mer has been

expressed in E. coli and shown to neutralize snake venoms. However, possible endotoxin

concerns warrant an investigation into other microbial hosts. The methylotrophic yeast,

Pichia pastoris, shows promise as an alternative host. Active LTNF peptide was

expressed and purified in P. pastoris. This was accomplished by subcloning a tandem

repeat of the first 15 N-terminal amino acids of lethal toxin neutralizing factor (LTNF-

15) into the Pichia expression vector, PPIC9K and transforming the yeast via

electroporation. Expression of LTNF-15 from Pichia was verified by applying a

fluorescent histidine tag stain onto a SDS-PAGE gel containing supernatant samples of P.

pastoris clones. Purification of the LTNF-15 peptide was conducted by Ni-NTA

purification. It was found that expressed LTNF-15 peptide demonstrated neutralizing

activity in an in vitro assay using azocasein. In addition, it was determined that a protein

concentration of 70 mg/L of LTNF-15 was attained during the fermentation process.

Thus, showing promise of Pichia as a viable production host.

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