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    • 7. 发明授权
    • Genetically encoded initiator for polymer growth from proteins
    • 用于聚合物从蛋白质生长的基因编码引发剂
    • US08816001B2
    • 2014-08-26
    • US13788710
    • 2013-03-07
    • Franklin and Marshall College
    • Ryan A. MehlKrzysztof MatyjaszewskiSaadyah Averick
    • A61K47/48C07C229/00C07C69/00C07C69/63C07C247/00C07K1/00
    • C12P21/00C07C229/36C07C231/14C07C233/54C07C233/81C07C247/10C07C247/12C07K14/001C07K14/43595C08F289/00C08F2438/01C12P21/02
    • This invention pertains to methods for producing homogeneous recombinant proteins that contain polymer initiators at defined sites. The unnatural amino acid, 4-(2′-bromoisobutyramido)phenylalanine of formula 1, was designed and synthesized as a molecule comprising a functional group further comprising an initiator for an atom-transfer radical polymerization (‘ATRP”) that additionally would provide a stable linkage between the protein and growing polymer. We evolved a Methanococcus jannaschii (Mj) tyrosyl-tRNA synthetase/tRNACUA pair to genetically encode this unnatural amino acid in response to an amber codon. To demonstrate the utility of this functional amino acid, we produced Green Fluorescent Protein with the unnatural amino acid initiator of formula 1 site-specifically incorporated on its surface (GFP-1). Purified GFP-1 was then used as an initiator under standard ATRP conditions with oligo(ethylene oxide)monomethyl ether methacrylate, efficiently producing a polymer-GFP bioconjugate wherein the polymer is connected at a specifically selected site on GFP.
    • 本发明涉及用于制备在限定位点含有聚合物引发剂的均质重组蛋白的方法。 设计并合成式1的非天然氨基酸4-(2'-溴异丁酰胺基)苯丙氨酸作为包含进一步包含原子转移自由基聚合引发剂(“ATRP”)的官能团的分子,其另外将提供 蛋白质与生长聚合物之间的稳定连接。 我们进化了一种甲烷球菌(Mj)酪氨酰-tRNA合成酶/ tRNACUA对以响应于琥珀密码子遗传编码该非天然氨基酸。 为了证明这种功能性氨基酸的实用性,我们用表面特异性引入(GFP-1)的式1的非天然氨基酸引发剂制备了绿色荧光蛋白。 然后将纯化的GFP-1用作标准ATRP条件下的引发剂,用寡聚(环氧乙烷)单甲醚甲基丙烯酸酯作为引发剂,有效生产聚合物-GFP生物缀合物,其中聚合物连接在GFP的特定选择位点。