Rational and efficient preparation of a chimeric protein containing a tandem dimer of thrombopoietin mimetic peptide fused to human growth hormone in Escherichia coli
Rational and efficient preparation of a chimeric protein containing a tandem dimer of thrombopoietin mimetic peptide fused to human growth hormone in Escherichia coli
复制标题
合理高效制备大肠杆菌中血小板生成素模拟肽与人生长激素融合的嵌合蛋白
DOI：
10.1007/s00253-012-4553-7
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发表时间：
2013-04-01
影响因子：
5
通讯作者：
Wang, Junping
中科院分区：
文献类型：
--
作者：
Wang, Song;Shen, Mingqiang;Wang, Junping
血小板生成素模拟肽（TMP），特别是以二聚体的形式，在体外显示出强大的巨核细胞生成活性。然而，通过基因工程的方法制备这种具有高生物活性的短肽是困难的。本研究利用可溶性表达技术在大肠杆菌中表达了一种嵌合蛋白，该蛋白含有TMP的串联二聚体（dTMP）和人生长激素（hGH），人生长激素是一种造血生长因子，激活与血小板生成素相同的信号通路。通过合理利用XmnI和EcoRV酶切位点，将dTMP-GH的PCR片段插入到质粒载体pMAL-p2 X中，位于Xa因子酶切位点后，与麦芽糖结合蛋白（MBP）基因符合读码框。在优化的条件下，获得了可溶性MBP-dTMP-GH融合蛋白的高效表达。采用淀粉树脂层析、Xa因子酶切、疏水层析和凝胶过滤等方法分离得到dTMP-GH融合蛋白。最后，通过高效液相色谱、质谱和氨基酸序列分析，获得了高纯度（> 98%）且无多余氨基酸的dTMP-GH融合蛋白。功能实验表明，dTMP-GH能促进小鼠骨髓巨核细胞的增殖和成熟，并呈剂量依赖性。此外，dTMP-GH对巨核细胞生成的作用比等摩尔浓度的dTMP和rhGH更强。本工作为以TMP为基础合成血小板生成剂提供了一条新的途径。
The 14-mer thrombopoietin mimetic peptide (TMP), especially in the form of dimer, displayed potent megakaryocytopoiesis activity in vitro. However, it is difficult to prepare such short peptide with high bioactivity through gene-engineering approaches. In this study, a chimeric protein containing a tandem dimer of TMP (dTMP) fused to human growth hormone (hGH), a kind of hematopoietic growth factor that activates the same signal pathways as thrombopoietin, was produced inEscherichia coliby soluble expression. By rational utilization of theXmnI andEcoRV restriction sites, a PCR fragment encoding dTMP-GH was inserted into the plasmid vector pMAL-p2X at the position right after Xa factor cleavage site, in frame with maltose-binding protein (MBP) gene. Under optimized conditions, a high-level expression of soluble MBP-dTMP-GH fusion protein was obtained. By application of amylose resin chromatography, Xa factor digestion, hydrophobic chromatography followed by gel filtration, the dTMP-GH fusion protein was separated. Finally, a relatively high yield of dTMP-GH fusion protein with high purity (>98 %) and without redundant amino acid was achieved, as identified by high-performance liquid chromatography, mass spectrometry, and amino acid sequencing. The functional assays showed that dTMP-GH could promote the proliferation of megakaryoblast cells and maturation of murine megakaryocytes derived from bone marrow, in a dose-dependent manner. Moreover, an enhanced effect of dTMP-GH on megakaryocytopoiesis was found as compared with equimolar concentration of dTMP and rhGH. This work provides a new avenue to generate thrombopoietic agents based on TMP.