Purification of GST-Tagged major capsid protein for aptamer-based detection of ranavirus
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 poses a significant threat to amphibian populations, necessitating rapid and reliable diagnostic tools. This study optimized protein purification methods for Ranavirus major capsid protein (MCP), which is the primary structural protein of the virus. The protein expression was conducted using a three-day protocol. We purified our protein using two different types of affinity chromatography resin: glutathione transferase (GST) and Ni-NTA. Protein concentration and purity was assessed using Nanodrop spectrophotometer (A260/A280 ratios) and SDS-PAGE gel electrophoresis. Initial purification with a GST-tag successfully yielded protein; however, Nanodrop analysis (A260/A280 > 1.24) revealed DNA contamination, with a protein concentration of less than 5 μM. Gel electrophoresis confirmed the presence of DNA contamination, requiring an alternative purification approach. An initial attempt to remove DNA using nuclease led to a significant loss of protein yield. To resolve this issue, a His-tag purification system was used instead of a GST-tag, successfully eliminating DNA contamination. This improvement allows us to use a cleaner Ranavirus major capsid protein sample for ouraptamer-binding studies and to avoid experimental complications that might result from the presence of the contaminating DNA.
Purification of GST-Tagged major capsid protein for aptamer-based detection of ranavirus
Ranavirus poses a significant threat to amphibian populations, necessitating rapid and reliable diagnostic tools. This study optimized protein purification methods for Ranavirus major capsid protein (MCP), which is the primary structural protein of the virus. The protein expression was conducted using a three-day protocol. We purified our protein using two different types of affinity chromatography resin: glutathione transferase (GST) and Ni-NTA. Protein concentration and purity was assessed using Nanodrop spectrophotometer (A260/A280 ratios) and SDS-PAGE gel electrophoresis. Initial purification with a GST-tag successfully yielded protein; however, Nanodrop analysis (A260/A280 > 1.24) revealed DNA contamination, with a protein concentration of less than 5 μM. Gel electrophoresis confirmed the presence of DNA contamination, requiring an alternative purification approach. An initial attempt to remove DNA using nuclease led to a significant loss of protein yield. To resolve this issue, a His-tag purification system was used instead of a GST-tag, successfully eliminating DNA contamination. This improvement allows us to use a cleaner Ranavirus major capsid protein sample for ouraptamer-binding studies and to avoid experimental complications that might result from the presence of the contaminating DNA.
Comments
Poster presentation at the 2025 Student Research Symposium.