Purification of sumo-tagged ranavirus major capsid protein

Presentation Type

Abstract

Faculty Advisor

Nina Goodey

Access Type

Event

Start Date

25-4-2025 12:00 PM

End Date

25-4-2025 1:00 PM

Description

Ranavirus has been detected in various amphibian species and it leads to infections and ultimately mortality in frogs. It is crucial to be able to identify and track the virus in natural and commercial environments, but the current testing methods have major drawbacks because they are intricate, costly, and time-consuming. This study optimized protein purification protocols to enable the development of a rapid field test for Ranavirus detection. The protein was initially expressed using a three-day protocol and purified via GST-tag affinity chromatography. This protocol outlines the expression of MCP-PGEX protein in E. coli, induction with IPTG, and collection of protein-rich pellets via centrifugation for storage at -80°C. Collected fractions were analyzed using a Nanodrop spectrophotometer to assess purity and concentration, monitoring absorbance at 280 nm (A280) and the A260/A280 ratio. High A280 values and low A260/A280 ratios indicate high protein yield with minimal DNA contamination, respectively. Wash 1 (samples 1-12) had high A260/A280 ratios. For example, the ratios in samples 8 (6.000) and 9 (7.524) indicated significant DNA contamination. These samples were unsuitable for downstream applications. Initial efforts to eliminate contamination using nucleases were only partially effective and significantly reduced protein concentration. To resolve this issue, we revised our purification protocol by adopting a His-tag system instead of a GST-tag. The altered procedure resulted in efficient purification of DNA-free protein, which is needed for aptamer binding assays.

Comments

Poster presentation at the 2025 Student Research Symposium.

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Apr 25th, 12:00 PM Apr 25th, 1:00 PM

Purification of sumo-tagged ranavirus major capsid protein

Ranavirus has been detected in various amphibian species and it leads to infections and ultimately mortality in frogs. It is crucial to be able to identify and track the virus in natural and commercial environments, but the current testing methods have major drawbacks because they are intricate, costly, and time-consuming. This study optimized protein purification protocols to enable the development of a rapid field test for Ranavirus detection. The protein was initially expressed using a three-day protocol and purified via GST-tag affinity chromatography. This protocol outlines the expression of MCP-PGEX protein in E. coli, induction with IPTG, and collection of protein-rich pellets via centrifugation for storage at -80°C. Collected fractions were analyzed using a Nanodrop spectrophotometer to assess purity and concentration, monitoring absorbance at 280 nm (A280) and the A260/A280 ratio. High A280 values and low A260/A280 ratios indicate high protein yield with minimal DNA contamination, respectively. Wash 1 (samples 1-12) had high A260/A280 ratios. For example, the ratios in samples 8 (6.000) and 9 (7.524) indicated significant DNA contamination. These samples were unsuitable for downstream applications. Initial efforts to eliminate contamination using nucleases were only partially effective and significantly reduced protein concentration. To resolve this issue, we revised our purification protocol by adopting a His-tag system instead of a GST-tag. The altered procedure resulted in efficient purification of DNA-free protein, which is needed for aptamer binding assays.