title: Expression and purification of coronavirus envelope proteins using a modified β-barrel construct.
abstract: Coronavirus envelope (E) proteins are short (~100 residues) polypeptides that contain at least one transmembrane (TM) domain and a cluster of 2-3 juxtamembrane cysteines. These proteins are involved in viral morphogenesis and tropism, and their absence leads in some cases to aberrant virions, or to viral attenuation. In common to other viroporins, coronavirus envelope proteins increase membrane permeability to ions. Although an NMR-based model for the TM domain of the E protein in the severe acute respiratory syndrome virus (SARS-CoV E) has been reported, structural data and biophysical studies of full length E proteins are not available because efficient expression and purification methods for these proteins are lacking. Herein we have used a novel fusion protein consisting of a modified β-barrel to purify both wild type and cysteine-less mutants of two representatives of coronavirus E proteins: the shortest (76 residues), from SARS-CoV E, and one of the longest (109 residues), from the infectious bronchitis virus (IBV E). The fusion construct was subsequently cleaved with cyanogen bromide and all polypeptides were obtained with high purity. This is an approach that can be used in other difficult hydrophobic peptides.
authors: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
authors: Krupakar
authors: K
authors: Parthasarathy
authors: Huang
authors: H
authors: Lu
authors: Wahyu
authors: W
authors: Surya
authors: Ardcharaporn
authors: A
authors: Vararattanavech
authors: Konstantin
authors: K
authors: Pervushin
authors: Jaume
authors: J
authors: Torres
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008051:Lipid Bilayers / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry / Q000235:genetics*; D045169:Severe Acute Respiratory Syndrome / Q000821:virology; D014461:Ultracentrifugation; D015854:Up-Regulation; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D008051:Lipid Bilayers; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins