Characterisation of a novel growth hormone variant comprising a thioether link between Cys182 and Cys189.
A novel variant of recombinant human growth hormone (r-hGH), isolated from biopharmaceutical preparations produced in E. coli, was identified and characterised. This variant contains a nonreducible thioether bridge near the C terminus between Cys182 and Cys189 and was characterised using various analytical techniques. As previous work by Cunningham and Wells (1993) highlighted the involvement of several residues in this part of the sequence in the binding and affinity of the molecule to its receptor, the presence of this modified intramolecular link may have important implications with regard to the biological behaviour of the molecule. Furthermore, as the conversion of a disulfide into a thioether was previously reported for a therapeutic monoclonal antibody (Tous et al., 2005), this may imply that disulfide bridges located in this part of the molecule have a generic susceptibility to thioether formation. This in turn is relevant to the biopharmaceutical industry for monitoring the integrity of disulfide bridges near the protein C terminus. The present study exhibits a state of the art physicochemical investigation for the unequivocal elucidation of a novel structure involving peptide mapping with mass spectrometry and de novo peptide sequencing. Changes in the higher order structure of the molecule were highlighted by near UV circular dichroism and molecular modelling.
Datola
Antonio
A
Analytical Development Centre of Expertise, Industria Farmaceutica Serono, Via di Valle Caia 22, 00040 Ardea, Roma, Italy.
Richert
Sophie
S
Bierau
Horst
H
Agugiaro
David
D
Izzo
Anna
A
Rossi
Mara
M
Cregut
David
D
Diemer
Hélène
H
Schaeffer
Christine
C
Van Dorsselaer
Alain
A
Giartosio
Carlo Emanuele
CE
Jone
Carl
C
D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D013006:Growth Hormone / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013440:Sulfides / Q000737:chemistry*
D011994:Recombinant Proteins; D013440:Sulfides; D013006:Growth Hormone; D003545:Cysteine