Hydroxylamine-induced cleavage of the asparaginyl-glycine motif in the production of recombinant proteins: the case of insulin-like growth factor I.
Hydroxylamine-induced cleavage at the asparaginyl-glycine dipeptide site inserted between the two moieties of recombinant fusion proteins has been used at both the analytical and the preparative scale to obtain the mature protein. In this study a model protein containing a fusion precursor of insulin-like growth factor I was used to investigate the influence of the operating conditions on the cleavage reaction and the formation of undesired side products such as hydroxamate and deamidated analogs. Moreover, the stability of the cleavage site toward deamidation was examined and a chemometric study performed to define the effect of the reaction conditions on the cleavage yield and on the formation of side products.
Antorini
M
M
Pharmacia & Upjohn, Nerviano, Italy.
Breme
U
U
Caccia
P
P
Grassi
C
C
Lebrun
S
S
Orsini
G
G
Taylor
G
G
Valsasina
B
B
Marengo
E
E
Todeschini
R
R
Andersson
C
C
Gellerfors
P
P
Gustafsson
J G
JG
D000595:Amino Acid Sequence; D004151:Dipeptides / Q000378:metabolism; D005285:Fermentation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006877:Hydroxamic Acids / Q000378:metabolism; D019811:Hydroxylamine / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis* / Q000302:isolation & purification; D007526:Isoelectric Point; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D018629:Spectrometry, Mass, Secondary Ion; D013885:Thiosulfates / Q000378:metabolism
D004151:Dipeptides; D006877:Hydroxamic Acids; D011993:Recombinant Fusion Proteins; D013885:Thiosulfates; D019811:Hydroxylamine; C017033:aspartylglycine; D007334:Insulin-Like Growth Factor I; D008715:Methionine; C017717:sodium thiosulfate