Extension of in vivo half-life of biologically active peptides via chemical conjugation to XTEN protein polymer
Extension of in vivo half-life of biologically active peptides via chemical conjugation to XTEN protein polymer
复制标题
DOI：
10.1093/protein/gzt048
复制
发表时间：
2013-11-01
影响因子：
2.4
通讯作者：
Schellenberger, Volker
中科院分区：
文献类型：
--
作者：
Podust, Vladimir N.;Sim, Bee-Cheng;Schellenberger, Volker
XTEN，非结构化生物可降解蛋白质，已被用于延长基因融合的治疗性蛋白质和肽的体内半衰期。为了将XTEN技术的应用扩展到其他类别分子的半衰期延长，开发了XTEN蛋白聚合物和化学XTEN化的方法。两种XTEN前体被工程化以含有酶促可去除的纯化标签。蛋白质容易在细菌中表达，并通过层析技术纯化至均一。作为原理证明，使用马来酰亚胺硫醇化学将GLP 2 -2G肽与两种XTEN蛋白聚合物中的每一种化学缀合。XTEN蛋白质聚合物的单分散性质使得能够使用反相高效液相色谱法（RP-HPLC）和电喷雾电离质谱法监测反应以及检测缀合状态下的肽修饰。通过制备型RP-HPLC将所得GLP 2 -2G-XTEN缀合物纯化至均匀。与重组融合GLP 2 -2G-XTEN相比，化学偶联GLP 2 -2G-XTEN分子在大鼠中表现出相当的体外活性、体外血浆稳定性和药代动力学。这些数据表明，化学XTEN化可以有效地延长广谱生物活性分子的半衰期，从而扩大其适用性。
XTEN, unstructured biodegradable proteins, have been used to extend the in vivo half-life of genetically fused therapeutic proteins and peptides. To expand the applications of XTEN technology to half-life extension of other classes of molecules, XTEN protein polymers and methods for chemical XTENylation were developed. Two XTEN precursors were engineered to contain enzymatically removable purification tags. The proteins were readily expressed in bacteria and purified to homogeneity by chromatography techniques. As proof-of-principle, GLP2-2G peptide was chemically conjugated to each of the two XTEN protein polymers using maleimidethiol chemistry. The monodisperse nature of XTEN protein polymer enabled reaction monitoring as well as the detection of peptide modifications in the conjugated state using reverse phase-high performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry. The resulting GLP2-2G-XTEN conjugates were purified by preparative RP-HPLC to homogeneity. In comparison with recombinantly fused GLP2-2G-XTEN, chemically conjugated GLP2-2G-XTEN molecules exhibited comparable in vitro activity, in vitro plasma stability and pharmacokinetics in rats. These data suggest that chemical XTENylation could effectively extend the half-life of a wide spectrum of biologically active molecules, therefore broadening its applicability.