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 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.
Cazalis
Chrystelle S
CS
Laboratory for Biomolecular Materials Research, Department of Surgery, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Haller
Carolyn A
CA
Sease-Cargo
Lisa
L
Chaikof
Elliot L
EL
D001665:Binding Sites / Q000502:physiology; D006801:Humans; D011092:Polyethylene Glycols / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D017384:Sequence Deletion; D018180:Thrombomodulin / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*
D018180:Thrombomodulin; D011092:Polyethylene Glycols