PURPOSE: N-terminal site-specific mono-PEGylation of recombinant human epidermal growth factor (EGF) was accomplished using polyethyleneglycol (PEG) derivatives (Mw = 2000 and 5000) through a reactive terminal aldehyde group. METHODS: The site-specific PEG conjugation was conducted ata slightly acidic pH condition (pH 5.5). The mono-PEGylation was targeted to an alpha-amine group at the N-terminal end of EGF to minimize reduction of biologic activity. Tryptic digestion mapping and MALDI-TOF MS techniques were applied to show the occurrence of mono-PEGylation at the N-terminus of EGF. RESULTS: The site-specific mono-PEGylated EGF, when compared with native EGF, fully retained its in vitro biologic activities such a cell proliferation and intracellular signal transduction. This revealed that although a synthetic polymer of a PEG was covalently conjugated to EGF, the internalized complex of PEGylated EGF-receptor within cells did not hamper the intracellular signal transduction events. The PEGylated EGF also exhibited a prolonged circulation in blood stream in vivo and markedly enhanced physical stability whe incubated with tissue homogenate. CONCLUSION: N-terminally mono-PEGylated EGF shows increased physical stability while retaining its biologic activity.
PURPOSE: N-terminal site-specific mono-PEGylation of recombinant humanepidermal growth factor (EGF) was accomplished using polyethyleneglycol (PEG) derivatives (Mw = 2000 and 5000) through a reactive terminal aldehyde group. METHODS: The site-specific PEG conjugation was conducted ata slightly acidic pH condition (pH 5.5). The mono-PEGylation was targeted to an alpha-amine group at the N-terminal end of EGF to minimize reduction of biologic activity. Tryptic digestion mapping and MALDI-TOF MS techniques were applied to show the occurrence of mono-PEGylation at the N-terminus of EGF. RESULTS: The site-specific mono-PEGylated EGF, when compared with native EGF, fully retained its in vitro biologic activities such a cell proliferation and intracellular signal transduction. This revealed that although a synthetic polymer of a PEG was covalently conjugated to EGF, the internalized complex of PEGylated EGF-receptor within cells did not hamper the intracellular signal transduction events. The PEGylated EGF also exhibited a prolonged circulation in blood stream in vivo and markedly enhanced physical stability whe incubated with tissue homogenate. CONCLUSION: N-terminally mono-PEGylated EGF shows increased physical stability while retaining its biologic activity.
Authors: Ryan G Soderquist; Erin D Milligan; Evan M Sloane; Jacqueline A Harrison; Klarika K Douvas; Joseph M Potter; Travis S Hughes; Raymond A Chavez; Kirk Johnson; Linda R Watkins; Melissa J Mahoney Journal: J Biomed Mater Res A Date: 2009-12 Impact factor: 4.396
Authors: Ryan G Soderquist; Erin D Milligan; Jacqueline A Harrison; Raymond A Chavez; Kirk W Johnson; Linda R Watkins; Melissa J Mahoney Journal: J Biomed Mater Res A Date: 2010-06-01 Impact factor: 4.396
Authors: Weiping Gao; Wenge Liu; J Andrew Mackay; Michael R Zalutsky; Eric J Toone; Ashutosh Chilkoti Journal: Proc Natl Acad Sci U S A Date: 2009-08-25 Impact factor: 11.205
Authors: Jun-Beom Park; Young Min Kwon; Tien-Yi Lee; Remy Brim; Mei-Chuan Ko; Roger K Sunahara; James H Woods; Victor C Yang Journal: J Control Release Date: 2009-10-24 Impact factor: 9.776