Affinity Chromatography Based on a Combinatorial Strategy for rErythropoietin Purification
Affinity Chromatography Based on a Combinatorial Strategy for rErythropoietin Purification
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DOI：
10.1021/co1000663
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发表时间：
2011-05-01
影响因子：
--
通讯作者：
Camperi, Silvia A.
中科院分区：
文献类型：
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作者：
Martinez-Ceron, Maria C.;Marani, Mariela M.;Camperi, Silvia A.
含有10个以下氨基酸的小肽是构建工业蛋白质纯化亲和层析系统的很有前途的候选配体。组合多肽合成策略的应用极大地促进了为任何给定的感兴趣的蛋白质寻找合适的配体。在这里，我们试图寻找与用于治疗贫血的重组人促红细胞生成素(RhEPO)有亲和力的多肽配体。用拆分-偶合-重组法在HMBA-ChemMatrix树脂上合成了一个含有八肽X-Phe-X-X-Ala-Gly的组合文库，其中X=Ala、Asp、Glu、Phe、His、Leu、Asn、Pro、Ser或Thr。在文库筛选中，将重组人促红细胞生成素与德克萨斯红或生物素偶联。分离出与链霉亲和素-过氧化物酶呈阳性反应的荧光珠或荧光珠。切割后，用基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOF MS)对多肽进行测序。57颗珠子呈阳性反应。合成了具有更多共识的多肽，并用等离子体共振生物传感器评价了它们与重组人促红细胞生成素的亲和力。得到了1-18微米范围内的离解常数值。最好的两种多肽被固定在Sepharose上，得到的层析基质与rhEPO具有亲和力，解离常数在1.8~2.7亩M之间。中国仓鼠卵巢(CHO)细胞培养上清液中加入rhEPO，人工混合物负载在多肽-Sepharose柱上。从洗脱部分回收重组人促红细胞生成素，得率为90%，纯度分别为95%和97%。
Small peptides containing fewer than 10 amino acids are promising ligand candidates with which to build affinity chromatographic systems for industrial protein purification. The application of combinatorial peptide synthesis strategies greatly facilitates the discovery of suitable ligands for any given protein of interest. Here we sought to identify peptide ligands with affinity for recombinant human erythropoietin (rhEPO), which is used for the treatment of anemia. A combinatorial library containing the octapeptides X-X-X-Phe-X-X-Ala-Gly, where X = Ala, Asp, Glu, Phe, His, Leu, Asn, Pro, Ser, or Thr, was synthesized on HMBA-ChemMatrix resin by the divide-couple-recombine method. For the library screening, rhEPO was coupled to either Texas Red or biotin. Fluorescent beads or beads showing a positive reaction with streptavidin-peroxidase were isolated. After cleavage, peptides were sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Fifty-seven beads showed a positive reaction. Peptides showing more consensuses were synthesized, and their affinity to rhEPO was assessed using a plasma resonance biosensor. Dissociation constant values in the range of 1-18 mu M were obtained. The best two peptides were immobilized on Sepharose, and the resultant chromatographic matrixes showed affinity for rhEPO with dissociation constant values between 1.8 and 2.7 mu M. Chinese hamster ovary (CHO) cell culture supernatant was spiked with rhEPO, and the artificial mixture was loaded on Peptide-Sepharose columns. The rhEPO was recovered in the elution fraction with a yield of 90% and a purity of 95% and 97% for PI-Sepharose and P2-Sepharose, respectively.