Master of Science (MS)
Claire . Komives
Antivenom, Lethal Toxin Neutralizing Factor, LTNF, Pichia Pastoris, Rattlesnake, Tandem Repeats
Chemical engineering; Biochemistry; Bioengineering
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.
Juarez Contreras, Israel, "Expression and Purification of Snake Antivenom Peptide in Pichia Pastoris" (2019). Master's Theses. 5066.