Site-directed pegylation of recombinant interleukin-2 at its glycosylation site.
We have modified recombinant interleukin-2 (rIL-2) to facilitate site-directed covalent attachment of monomethoxy polyethylene glycol (PEG). The site chosen for modification and subsequent covalent attachment with PEG (PEGylation) was the single glycosylation position found in the native interleukin-2 (IL-2). The mutant protein was expressed in E. coli, purified, and PEGylated with a PEG-maleimide reagent to obtain PEG-cys3-rIL-2. The PEG-cys3-rIL-2 had full bioactivity relative to the unmodified molecule and had an increase in hydrodynamic size sufficient to increase its systemic exposure by approximately 4 fold. This method has general applicability for modifying any therapeutic protein at a specific site and thereby alter its potency. In particular, it can be used to attach PEG to prokaryotically expressed recombinant proteins at their glycosylation sites.
Cetus Corporation, Emeryville, CA 94608.
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RJ
Goodson
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NV
Katre
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006031:Glycosylation; D007376:Interleukin-2 / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011092:Polyethylene Glycols / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D007376:Interleukin-2; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; C028210:monomethoxypolyethylene glycol; D003545:Cysteine