Developing a yeast based screening system for sea anemone actinoporins

Author: Ly Kim

Kim, Ly, 2021 Developing a yeast based screening system for sea anemone actinoporins, Flinders University, College of Medicine and Public Health

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Type II cytolysins, also known as actinoporins, are abundant in sea anemone venom. Due to their ability to form a cation-selective conduction pathway which allows ions or molecules to move in and out of cells to make an osmotic imbalance which can lead to cell death, actinoporins are of great interest for development as cancer therapeutics. This research firstly aimed to validate the partial actinoporin sequence from the sea anemone Entacmae quadricolor. Secondly, the research aimed to develop and assess a Saccharomyces cerevisiae yeast screening system for evaluating actinoporins from E. quadricolor and other anemone species and to compare this assay to the established haemolysis assay. This research hypothesized that yeast would be an effective system that could be used for screening potential actinoporins for development as anti-cancer therapeutic drugs. Multiple PCR products representing the E. quadricolor gene were obtained and purified. Sanger sequencing of these products was not successful, but this was due to the PCR products containing more than one sequence/gene. A Blast search of the newly assembled E. quadricolor transcriptome identified the possible presence of 14 highly similar actinoporin genes, some of which contain multiple isoforms. Venoms from E. quadricolor were collected at three time points, before hosting a Pairs of Amphiprion percula anemonefish, four weeks after hosting the fish, and four weeks after the fish were removed. These venoms were used to test the haemolysis and yeast screening assay. The sheep erythrocyte haemolysis assay showed that all venom samples tested at concentration 0.5 μg/mL to 8 μg/mL could lyse the cells but there was a lot of variation in EC50 at 415 nm of samples collected at the three time points tested. The yeast assay was developed using two different wild type Saccharomyces cerevisiae strains, BY4741 and Invsc2. Anemone venom was able to inhibit yeast growth up to 61% in a dose dependent manner at protein concentrations of 100 µg/mL. However, the yeast assay was unable to discriminate between the potency of the different venoms in the same manner as the hemolysis assay. The determination of the E. quadricolor genome and cloning of individual actinoporin genes products is required to validate the number of actinoporin genes expressed in the E. quadricolor transcriptome. The variability of the results observed in the haemolysis assay could be improved if a method for fractionating and obtaining a pure 15- 20 kDa fraction were developed using HLPC. Once a purer actinoporin containing fraction is obtained from the venom, it could be used in the yeast assay to determine the role of the actinoporin in slowing yeast growth. Hence, the yeast screening assay could be used for screening actinoporins for development as anticancer drugs.

Keywords: Sea anemone, Multigene families, Venom, Yeast Screening, High throughput screening, anticancer drugs

Subject: Biotechnology thesis

Thesis type: Masters
Completed: 2021
School: College of Medicine and Public Health
Supervisor: Professor Catherine Abbott