A novel solid-phase site-specific PEGylation enhances the in vitro and in vivo biostabilty of recombinant human keratinocyte growth factor 1.
A novel solid-phase site-specific PEGylation enhances the in vitro and in vivo biostabilty of recombinant human keratinocyte growth factor 1.
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新型固相位点特异性聚乙二醇化增强重组人角质形成细胞生长因子 1 的体外和体内生物稳定性
DOI：
10.1371/journal.pone.0036423
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Li X
中科院分区：
文献类型：
--
作者：
Huang Z;Zhu G;Sun C;Zhang J;Zhang Y;Zhang Y;Ye C;Wang X;Ilghari D;Li X
角质细胞生长因子1（KGF-1）已被证明可用于治疗与皮肤附件、肝脏、肺和胃肠道疾病相关的病理。然而，该蛋白质的稳定性差和血浆半衰期短限制了其治疗应用。虽然使用溶液相PEG化可以改善重组人KGF-1（rhKGF-1）的稳定性并延长其循环半衰期，但由于多次和/或不受控制的PEG化，这些制剂具有异质结构并且通常具有低的比活性。在本研究中，采用一种新的固相聚乙二醇化策略来生产均一的单聚乙二醇化rhKGF-1。将rhKGF-1蛋白固定在Heparin-Sepharose柱上，然后通过在蛋白的N-末端氨基酸处的还原性烷基化进行位点选择性聚乙二醇化反应。单聚乙二醇化rhKGF-1经SP Sepharose离子交换层析纯化至均一，占PEG化反应所用rhKGF-1总量的40%以上。我们的生物物理和生物化学研究表明，固相聚乙二醇化显着提高了在体外和体内的生物稳定性，而不影响整个蛋白质的结构。此外，药代动力学分析表明，修饰的rhKGF-1具有相当长的血浆半衰期比其完整的对应物。我们的细胞基础分析表明，与rhKGF-1类似，PEG化rhKGF-1通过激活MAPK/Erk通路诱导NIH 3 T3细胞增殖。值得注意的是，与rhKGF-1相比，PEG化rhKGF-1在大鼠中对CCl 4诱导的损伤表现出更大的肝保护作用。
Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the in vitro and in vivo biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl4-induced injury in rats compared to rhKGF-1.
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影响因子：
3.8
作者：
CHEN, BL;ARAKAWA, T;CHIEN, SL
通讯作者：
CHIEN, SL
影响因子：
4.1
作者：
Cai, Yongqing;Yue, Peng
通讯作者：
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影响因子：
1.6
作者：
Luo, YD;Cho, HH;McKeehan, WL
通讯作者：
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4.7
作者：
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通讯作者：
Filpula, David
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3.7
作者：
Lee, H;Jang, IH;Park, TG
通讯作者：
Park, TG