Research Article: Genetic PEGylation

Date Published: November 8, 2012

Publisher: Public Library of Science

Author(s): Seiichi Tada, Takashi Andou, Takehiro Suzuki, Naoshi Dohmae, Eiry Kobatake, Yoshihiro Ito, Nediljko Budisa.


Polyethylene glycol (PEG) was genetically incorporated into a polypeptide. Stop-anticodon-containing tRNAs were acylated with PEG-containing amino acids and were then translated into polypeptides corresponding to DNA sequences containing the stop codons. The molecular weights of the PEG used were 170, 500, 700, 1000, and 2000 Da, and the translation was confirmed by mass spectrometry. The PEG incorporation ratio decreased as the molecular weight of PEG increased, and PEG with a molecular weight of 1000 Da was only slightly incorporated. Although improvement is required to increase the efficiency of the process, this study demonstrates the possibility of genetic PEGylation.

Partial Text

Synthetic polymer–protein hybrids have been developed for use as therapeutic proteins or bioreactor enzymes [1]–[10]. Polyethylene glycol (PEG), which is nontoxic, nonimmunogenic, highly soluble in water, and approved by the U.S. Food and Drug Administration (FDA), is very useful in the preparation of therapeutic proteins [11], [12]. Some proteins trigger immune reactions, and proteases and other compounds inside the body can rapidly degrade them and remove them from the body. Many PEGylated protein drugs have been developed since the pioneering work of Abuchowsky et al. on the PEGylation of proteins [13], [14]. Although protein PEGylation has proven very valuable, many of the first-generation PEGylation products suffered from a severe loss in bioactivity [5]. This reduction in activity was mainly attributed to the chain lengths of the attached polymers and the site on the protein to which they are coupled. To overcome this difficulty, a more specific modification was achieved by exploiting the difference in the pKa value of the amine in the lysine side chain or the replacement of lysine residues with other amino acids [15]–[18]. The thiol groups in cysteine residues, the phenol groups of tyrosines, the amide groups of glutamines, or the incorporated His tag have also been used for this specific modification [19]–[29]. The site-specific polymer attachment has also been achieved with the complete synthetic construction of an erythropoietic protein [30], [31], consisting of an α-amino acid polypeptide chain of 166 residues by using native chemical ligation. Recent development of ligation methods has significantly expanded the possibility of bioorthogonal chemistry [32]–[36].



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