Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA.
Proc Natl Acad Sci U S A. 2011 Feb 22;108(8):3169-74. doi: 10.1073/pnas.1016863108. Epub 2011 Feb 4.
Recombinant protein therapeutics often suffer from short circulating half-life and poor stability, necessitating multiple injections and resulting in limited shelf-life. Conjugation to polyethylene glycol chains (PEG) extends the circulatory half-life of many proteins, but the methods for attachment often lack specificity, resulting in loss of biological activity. Using four-helix bundle cytokines as an example, we present a general platform that uses sortase-mediated transpeptidation to facilitate site-specific attachment of PEG to extend cytokine half-life with full retention of biological activity. Covalently joining the N and C termini of proteins to obtain circular polypeptides, again executed using sortase, increases thermal stability. We combined both PEGylation and circularization by exploiting two distinct sortase enzymes and the use of a molecular suture that allows both site-specific PEGylation and covalent closure. The method developed is general, uses a set of easily accessible reagents, and should be applicable to a wide variety of proteins, provided that their termini are not involved in receptor binding or function.
重组蛋白治疗药物通常半衰期短，稳定性差，需要多次注射，导致保质期有限。与聚乙二醇（PEG）链的缀合可以延长许多蛋白质的循环半衰期，但连接的方法往往缺乏特异性，导致生物活性丧失。我们以四螺旋束细胞因子为例，提出了一个通用平台，该平台使用 sortase 介导的转肽作用来促进 PEG 的定点附着，从而延长细胞因子的半衰期，同时保持其生物活性。通过使用 sortase 将蛋白质的 N 和 C 末端共价连接以获得环状多肽，也可以提高热稳定性。我们通过利用两种不同的 sortase 酶和使用分子缝线来同时进行 PEG 化和环化，该分子缝线允许定点 PEG 化和共价封闭。所开发的方法具有通用性，使用了一组易于获得的试剂，并且应该适用于广泛的蛋白质，只要它们的末端不参与受体结合或功能即可。
