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Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System
Published on: July 17, 2021
Expression and purification of MERS-CoV envelope protein, an essential viroporin, using the baculovirus expression
Entedar Alsaadi1, Dhafer Alghezi1, Ian Jones2
1Department of Microbiology, College of Medicine, University of Thi-Qar, Thi-Qar, Iraq.
Background And Objectives:
The causative agent of Middle East Respiratory Syndrome (MERS) is a zoonotic Coronavirus (MERS-CoV) identified in Saudi Arabia in 2012. The envelope (E) protein of MERS-CoV is a small viral protein which plays several essential roles during virus replication. To facilitate study of the structure and function of the E protein, recombinant MERS-CoV E protein was expressed using the baculovirus expression system.
Materials And Methods:
A recombinant E open reading frame including an 8-histidine tag at the amino terminus was designed and cloned into a baculovirus transfer vector. Following construction of a recombinant virus insect cells were infected and the expression of the E protein assessed by SDS-PAGE and Western blotting.
Results:
Recombinant E protein, tagged at the N-terminus with a polyhistidine sequence, with a molecular mass of 10.18 kD was identified by Western blotting with an anti-His antibody. Following large scale infection E protein was released by detergent mediated lysis of infected cells and purified by Immobilized Metal Ion Affinity Chromatography (IMAC).
Conclusion:
Purified full length recombinant MERS-CoV E protein can be isolated by IMAC and is suitable for further functional, biophysical or immunological studies.
Insights
Researchers successfully produced recombinant Middle East Respiratory Syndrome (MERS) envelope (E) protein using a baculovirus system. This purified MERS-CoV E protein is now available for further structural and functional studies.
Area of Science:
- Virology
- Molecular Biology
- Protein Expression
Background:
- Middle East Respiratory Syndrome (MERS) is caused by a zoonotic Coronavirus (MERS-CoV), first identified in Saudi Arabia in 2012.
- The MERS-CoV envelope (E) protein is crucial for viral replication.
- Understanding the MERS-CoV E protein's structure and function is vital for developing therapeutic strategies.
Purpose of the Study:
- To express and purify recombinant MERS-CoV E protein for further research.
- To facilitate structural and functional investigations of the MERS-CoV E protein.
Main Methods:
- A recombinant MERS-CoV E gene with an 8-histidine tag was cloned into a baculovirus transfer vector.
- Insect cells were infected with the recombinant virus to express the E protein.
- Protein expression was confirmed using SDS-PAGE and Western blotting, followed by purification via Immobilized Metal Ion Affinity Chromatography (IMAC).
Main Results:
- Recombinant MERS-CoV E protein, N-terminally tagged with a polyhistidine sequence, was successfully expressed and identified.
- The purified protein has a molecular mass of 10.18 kD.
- The protein was effectively purified using IMAC after detergent-mediated lysis of infected cells.
Conclusions:
- Full-length recombinant MERS-CoV E protein can be isolated using IMAC.
- The purified protein is suitable for subsequent functional, biophysical, and immunological studies.
- This work provides a foundation for deeper investigation into MERS-CoV pathogenesis and potential interventions.
