C-Terminal Site-Specific PEGylation of a Truncated Thrombomodulin Mutant with Retention of Full Bioactivity

Addition of poly(ethylene glycol) to bioactive proteins (PEGylation) improves their plasma half-life, enhances stability against proteolytic cleavage, and may also decrease protein immunogenicity. Characteristically, PEGylation usually involves a reaction to available lysine amino groups, some of wh...

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Bibliographic Details
Published in:Bioconjugate chemistry 2004-09, Vol.15 (5), p.1005-1009
Main Authors: Cazalis, Chrystelle S, Haller, Carolyn A, Sease-Cargo, Lisa, Chaikof, Elliot L
Format: Article
Language:English
Subjects:
Binding Sites - physiology
Biochemistry
Chemical synthesis
Humans
Polyethylene glycol
Polyethylene Glycols - analysis
Polyethylene Glycols - chemistry
Polyethylene Glycols - metabolism
Proteins
Sequence Deletion
Thrombomodulin - analysis
Thrombomodulin - genetics
Thrombomodulin - metabolism
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Summary:Addition of poly(ethylene glycol) to bioactive proteins (PEGylation) improves their plasma half-life, enhances stability against proteolytic cleavage, and may also decrease protein immunogenicity. Characteristically, PEGylation usually involves a reaction to available lysine amino groups, some of which may be within or near a bioactive site. Thus, most protocols are nonspecific and result in a loss of protein activity. We report herein a strategy for site-specific PEGylation of a thrombomodulin (TM) derivative at the C terminus. A truncated TM mutant consisting of epidermal growth factor (EGF)-like domains 4−6 was expressed in Escherichia coli with a C-terminal azido-methionine. The TM mutant was site-specifically conjugated to a methyl-PEG-triarylphosphine compound via the Staudinger reaction. Enzymatic activity of the TM construct before and after PEGylation was unchanged, which confirms the utility of this site-specific PEGylation scheme.
ISSN:1043-1802
1520-4812
DOI:10.1021/bc049903y