Synthesis and characterization of Respiratory Syncytial Virus protein G related peptides containing two disulfide bridges.
Respiratory Syncytial Virus (RSV) is the most important cause of bronchiolitis and viral pneumonia in infants and young children. Approximately 100000 children are hospitalized in the USA each year as a result of RSV infections. During the research and development of subunit human Respiratory Syncytial Virus vaccines (hRSV), we have produced numerous synthetic peptides and recombinant proteins containing the four cysteines of the highly conserved central region of the G attachment protein. For several of these disulfide-containing peptides, all possible oxidized isomers were synthesized using various oxidation conditions and resulting in different ratios of isomers. Each isolated isomer was fully characterized by RP-HPLC, FZCE and ES-MS after purification by preparative RP-HPLC. The different cysteine pairings were unambiguously established after enzymatic digestion, LC-MS analysis and peptide microsequencing. These synthesis and analytical methods were developed for the characterization on one hand, of recombinant fusion protein BBG2Na which is currently being investigated in advanced clinical phases as a very promising vaccine candidate, and on the other hand, for peptides which were synthesized to be evaluated as conjugate vaccines or as immunochemical tools, after covalent coupling to carrier proteins. Furthermore, these studies allowed us to determine which of the different possible isomers was the most stable and probably the preferred form in native conditions. Finally, the different oxidation and analysis conditions, should be useful for disulfide pairing studies of other peptides and proteins having the same 'xCxxCxxxxxCxxxCx' framework, such as G proteins of non-human RSV strains, developed by other groups as veterinary vaccine candidates for example.
Beck
A
A
Department of Physical-Chemistry, Center d'Immunologie Pierre Fabre, Saint-Julien-en-Genevois, France. alain.beck@pierre-fabre.com
Zorn
N
N
Bussat
M C
MC
Haeuw
J F
JF
Corvaïa
N
N
Nguyen
T N
TN
Bonnefoy
J Y
JY
Van Dorsselaer
A
A
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004220:Disulfides / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005976:Glutaral / Q000737:chemistry; D015707:HN Protein; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000737:chemistry; D012136:Respiratory Syncytial Viruses; D013820:Thermolysin / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism
D004220:Disulfides; D015707:HN Protein; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C116204:attachment protein G; D014357:Trypsin; D013820:Thermolysin; D005976:Glutaral