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Use of trypsin for rapid and efficient purification of murine sarcoma and leukemia virus
Abstract:
A method is described for purification of MSV-MuLV from culture supernatant of chronically infected 78A,1 rat embryo cell line. This method involves direct polyethylene glycol-NaCl precipitation of the low speed supernatant of culture fluid followed by digestion of the pellet with trypsin. This procedure efficiently disrupts large aggregates which normally entrap most of the virus. Highly purified virus can be obtained in very good yield by a combination of sedimentation velocity and isopycnic centrifugation : yields up to 100 A280 units (17 mg of protein) of purified virus per liter of culture fluid can be observed. This procedure appears well suited for large scale isolation of virion associated enzymatic activities.
Insights
This study presents an efficient method for purifying Moloney murine sarcoma virus-murine leukemia virus (MSV-MuLV) from cell culture. The technique uses polyethylene glycol precipitation and trypsin digestion, yielding highly purified virus for enzymatic studies.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Murine sarcoma virus-murine leukemia virus (MSV-MuLV) is a retrovirus often found in complex infections.
- Efficient purification of MSV-MuLV is crucial for studying its associated enzymatic activities.
- Existing methods may struggle with large viral aggregates present in culture supernatants.
Purpose of the Study:
- To develop an optimized method for the large-scale purification of MSV-MuLV.
- To improve the yield and purity of isolated MSV-MuLV.
- To facilitate the study of virion-associated enzymatic activities.
Main Methods:
- Direct polyethylene glycol-NaCl precipitation of low-speed supernatant from infected rat cell cultures.
- Trypsin digestion of the precipitated pellet to disrupt viral aggregates.
- Purification using a combination of sedimentation velocity and isopycnic centrifugation.
Main Results:
- The method effectively disrupts large aggregates entrapping virus particles.
- High yields of purified MSV-MuLV were achieved, up to 100 A280 units (17 mg protein) per liter of culture fluid.
- The procedure is suitable for large-scale isolation of purified virus.
Conclusions:
- This novel purification method significantly enhances MSV-MuLV recovery and purity.
- The optimized protocol is well-suited for large-scale purification of MSV-MuLV.
- The purified virus is ideal for investigating associated enzymatic functions.