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首页> 外文期刊>Protein Expression and Purification >Increasing the homogeneity, stability and activity of human serum albumin and interferon-alpha 2b fusion protein by linker engineering
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Increasing the homogeneity, stability and activity of human serum albumin and interferon-alpha 2b fusion protein by linker engineering

机译：通过接头工程提高人血清白蛋白和干扰素-α2b融合蛋白的均一性，稳定性和活性

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摘要

Previous studies in our laboratory have shown that when the N-terminus of interferon-alpha 2b (IFN-alpha 2b) was directly fused of to the C-terminus of human serum albumin (HSA), the resultant fusion protein (HSA-IFN-alpha 2b) was heterogeneous (migrated as doublets on non-reducing SDS-PAGE) and unstable (prone to form covalent aggregates). The heterogeneity and instability of HSA-IFN-alpha 2b was ascribed to the structural disturbance between HSA and IFN-alpha 2b. To alleviate such structural disturbance, linkers with different lengths (1, 2, 5, 10 amino acid residues) or different conformation (flexible linker (FL, GGGGS), rigid linker (RL, PAPAP) or helix-forming linker (HL, AEAAAKEAAAKA)) were inserted between HSA and IFN-alpha 2b. It was demonstrated that linker with 5 amino acid residues was sufficient to separated HSA and IFN-alpha 2b effectively, as fusion protein with this linker migrated as single band on non-reducing SDS-PAGE. The fusion proteins with FL, RL and HL linkers were purified to homogeneity with yields of 20%, while the recovery rate of HSA-IFN-alpha 2b was only 10%. Accelerated thermal stress tests showed that in contrast to HSA-IFN-alpha 2b, fusion proteins with FL, RL and HL linkers were free of aggregates after stored at 37 degrees C for 10 days. Stability tests also revealed that fusion proteins with FL, RL and HL linkers had different susceptibility to hydrolysis, with HSA-RL-IFN-alpha 2b being the least susceptible to hydrolysis at pH 6 and 7. Activity assay revealed that the insertion of FL, RL and HL linkers increased the anti-viral activity of fusion protein by 39%, 68% and 115%, respectively. (C) 2008 Elsevier Inc. All rights reserved.
机译：我们实验室先前的研究表明，当干扰素-α2b的N端（IFN-alpha 2b）直接与人血清白蛋白（HSA）的C端融合时，产生的融合蛋白（HSA-IFN- α2b）是异质的（在非还原性SDS-PAGE上以双峰形式迁移）且不稳定（易于形成共价聚集体）。 HSA-IFN-α2b的异质性和不稳定性归因于HSA和IFN-α2b之间的结构干扰。为减轻这种结构干扰，可使用不同长度（1、2、5、10个氨基酸残基）或不同构象的连接子（柔性连接子（FL，GGGGS），刚性连接子（RL，PAPAP）或螺旋形成的连接子（HL，AEAAAKEAAAKA） ））插入HSA和IFN-α2b之间。已经证明具有5个氨基酸残基的接头足以有效地分离HSA和IFN-α2b，因为具有该接头的融合蛋白在非还原性SDS-PAGE上以单条带迁移。具有FL，RL和HL接头的融合蛋白被纯化至同质，产率为20％，而HSA-IFN-α2b的回收率仅为10％。加速的热应力测试表明，与HSA-IFN-α2b相比，具有FL，RL和HL接头的融合蛋白在37°C保存10天后没有聚集体。稳定性测试还显示，带有FL，RL和HL接头的融合蛋白对水解的敏感性不同，HSA-RL-IFN-α2b在pH 6和7下最不易水解。活性分析表明，插入了FL， RL和HL接头分别使融合蛋白的抗病毒活性提高了39％，68％和115％。 （C）2008 Elsevier Inc.保留所有权利。

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