Expression and purification of coronavirus envelope proteins using a modified β-barrel construct

Area of Science:

• Biochemistry
• Structural Biology
• Virology

Background:

• Coronavirus envelope (E) proteins are short polypeptides crucial for viral morphogenesis and tropism.
• These viroporins increase membrane permeability, but structural studies are hindered by purification challenges.
• Previous research provided an NMR model for SARS-CoV E's transmembrane domain only.

Purpose of the Study:

• To develop efficient expression and purification methods for full-length coronavirus E proteins.
• To enable structural and biophysical characterization of E proteins from SARS-CoV and IBV.
• To establish a versatile approach for purifying other challenging hydrophobic peptides.

Main Methods:

• A novel fusion protein strategy using a modified beta-barrel was employed.
• Purification of both wild-type and cysteine-less mutants of SARS-CoV E and IBV E proteins.
• Cleavage of the fusion construct with cyanogen bromide to isolate target polypeptides.

Main Results:

• High purity was achieved for both wild-type and mutant coronavirus E proteins.
• The fusion protein approach successfully purified E proteins from SARS-CoV and IBV.
• Demonstrated the utility of this method for other difficult-to-purify hydrophobic peptides.

Conclusions:

• A novel and effective fusion protein method has been established for purifying coronavirus E proteins.
• This technique overcomes previous limitations in obtaining sufficient quantities of E proteins for study.
• The developed approach has broad applicability for the structural and biophysical investigation of hydrophobic viral peptides.