Expression and Purification of Coronavirus Envelope Proteins Using a Modified β-barrel Construct

Overview

Journal Protein Expr Purif

Specialty Molecular Biology

Date 2012 Jul 24

PMID 22819936

Citations 25

Authors

Krupakar Parthasarathy

Huang Lu

Wahyu Surya

Ardcharaporn Vararattanavech

Konstantin Pervushin

Jaume Torres

Affiliations

Soon will be listed here.

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.

Citing Articles

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Dimeric Transmembrane Structure of the SARS-CoV-2 E Protein.

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References

Maupetit J, Derreumaux P, Tuffery P . PEP-FOLD: an online resource for de novo peptide structure prediction. Nucleic Acids Res. 2009; 37(Web Server issue):W498-503. PMC: 2703897. DOI: 10.1093/nar/gkp323. View

Lopez L, Riffle A, Pike S, Gardner D, Hogue B . Importance of conserved cysteine residues in the coronavirus envelope protein. J Virol. 2008; 82(6):3000-10. PMC: 2258990. DOI: 10.1128/JVI.01914-07. View

Mortola E, Roy P . Efficient assembly and release of SARS coronavirus-like particles by a heterologous expression system. FEBS Lett. 2004; 576(1-2):174-8. PMC: 7126153. DOI: 10.1016/j.febslet.2004.09.009. View

Laue T, Stafford 3rd W . Modern applications of analytical ultracentrifugation. Annu Rev Biophys Biomol Struct. 1999; 28:75-100. DOI: 10.1146/annurev.biophys.28.1.75. View

Kochendoerfer G, Salom D, Lear J, Kent S, Degrado W . Total chemical synthesis of the integral membrane protein influenza A virus M2: role of its C-terminal domain in tetramer assembly. Biochemistry. 1999; 38(37):11905-13. DOI: 10.1021/bi990720m. View

Ye Y, Lee H, Yang W, Yang J . Calcium and lanthanide affinity of the EF-loops from the C-terminal domain of calmodulin. J Inorg Biochem. 2005; 99(6):1376-83. DOI: 10.1016/j.jinorgbio.2005.03.011. View

Gross E, WITKOP B . Nonenzymatic cleavage of peptide bonds: the methionine residues in bovine pancreatic ribonuclease. J Biol Chem. 1962; 237:1856-60. View

Kuo L, Hurst K, Masters P . Exceptional flexibility in the sequence requirements for coronavirus small envelope protein function. J Virol. 2006; 81(5):2249-62. PMC: 1865940. DOI: 10.1128/JVI.01577-06. View

Bertini I, Gelis I, Katsaros N, Luchinat C, Provenzani A . Tuning the affinity for lanthanides of calcium binding proteins. Biochemistry. 2003; 42(26):8011-21. DOI: 10.1021/bi034494z. View

10.

Baudoux P, Carrat C, Besnardeau L, Charley B, Laude H . Coronavirus pseudoparticles formed with recombinant M and E proteins induce alpha interferon synthesis by leukocytes. J Virol. 1998; 72(11):8636-43. PMC: 110275. DOI: 10.1128/JVI.72.11.8636-8643.1998. View

11.

BOS E, Luytjes W, van der Meulen H, Koerten H, Spaan W . The production of recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus. Virology. 1996; 218(1):52-60. PMC: 7130547. DOI: 10.1006/viro.1996.0165. View

12.

Nagano N, Ota M, Nishikawa K . Strong hydrophobic nature of cysteine residues in proteins. FEBS Lett. 1999; 458(1):69-71. DOI: 10.1016/s0014-5793(99)01122-9. View

13.

Ramjeesingh M, Huan L, Garami E, Bear C . Novel method for evaluation of the oligomeric structure of membrane proteins. Biochem J. 1999; 342 ( Pt 1):119-23. PMC: 1220444. View

14.

Park T, Kim J, Choi S, Kim Y . Cloning, expression, isotope labeling, purification, and characterization of bovine antimicrobial peptide, lactophoricin in Escherichia coli. Protein Expr Purif. 2009; 65(1):23-9. DOI: 10.1016/j.pep.2008.12.009. View

15.

Pautsch A, Schulz G . Structure of the outer membrane protein A transmembrane domain. Nat Struct Biol. 1998; 5(11):1013-7. DOI: 10.1038/2983. View

16.

Liao Y, Yuan Q, Torres J, Tam J, Liu D . Biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein. Virology. 2006; 349(2):264-75. PMC: 7111751. DOI: 10.1016/j.virol.2006.01.028. View

17.

Bauer C, Pinto L, Cross T, Lamb R . The influenza virus M2 ion channel protein: probing the structure of the transmembrane domain in intact cells by using engineered disulfide cross-linking. Virology. 1999; 254(1):196-209. DOI: 10.1006/viro.1998.9552. View

18.

Curtis K, Yount B, Baric R . Heterologous gene expression from transmissible gastroenteritis virus replicon particles. J Virol. 2002; 76(3):1422-34. PMC: 135785. DOI: 10.1128/jvi.76.3.1422-1434.2002. View

19.

Yuan Q, Liao Y, Torres J, Tam J, Liu D . Biochemical evidence for the presence of mixed membrane topologies of the severe acute respiratory syndrome coronavirus envelope protein expressed in mammalian cells. FEBS Lett. 2006; 580(13):3192-200. PMC: 7094218. DOI: 10.1016/j.febslet.2006.04.076. View

20.

Gorbalenya A, Snijder E, Spaan W . Severe acute respiratory syndrome coronavirus phylogeny: toward consensus. J Virol. 2004; 78(15):7863-6. PMC: 446116. DOI: 10.1128/JVI.78.15.7863-7866.2004. View