重组人Neuritin蛋白复性优化及初步结构表征的探究

Exploration of renaturation optimization and preliminary structural characterization of recombinant human Neuritin protein

摘要

目的 通过蛋白质的变性-复性策略及活性鉴定,探讨重组人Neuritin蛋白的天然结构。方法 1)重组蛋白的表达、结构及活性鉴定:按照毕赤酵母操作手册完成重组子的诱导表达,经疏水层析完成蛋白纯化;利用Western blot鉴定其免疫原性,还原电泳鉴定其纯度,非还原电泳和分子排阻色谱鉴定其结构;利用课题组已建立的CCK8法测定细胞存活率来评价Neuritin蛋白活性。2)蛋白变性:选择变性剂尿素和β-巯基乙醇,分别探讨变性剂浓度、变性时间和蛋白浓度对重组Neuritin蛋白变性的影响,经结构鉴定和活性检测,确定变性条件。3)蛋白复性:首先使用Injection buffer减少蛋白分子间的非特异性聚集;然后应用复性工作液,让蛋白自发地按照热力学最低能态进行重折叠。根据蛋白结构稳定性和活性恢复,确定复性条件。结果 1)诱导表达的Neuritin蛋白经疏水层析纯化后,Western blot确定其免疫学活性,还原电泳鉴定纯度为95%以上,非还原电泳的初步结构鉴定发现,该重组蛋白以二聚体为主,单体占比约31.37%。2)分别观察尿素及β-巯基乙醇的浓度、变性时间和蛋白浓度对重组Neuritin蛋白变性的影响,最终确定了Neuritin蛋白完全变性的条件为10 mol·L^-1尿素、10 mmol·L^-1 β-巯基乙醇作用于0.4 μg·uL^-1的重组Neuritin蛋白2 h。经进一步的分子排阻色谱实验和活性鉴定证实,在此条件下,蛋白空间结构充分去折叠、且活性完全丧失。3)蛋白复性结果显示,蛋白复性12~36 h,其结构趋于恒定,呈现稳定的“以单体为主,含有少量的二聚体”的结构形式;复性24 h,蛋白活性达到高峰。以此确定的蛋白复性条件,比较复性蛋白与变性前初始蛋白,活性检测显示,复性后的重组Neuritin蛋白活性升高;分子排阻色谱实验的结构鉴定结果显示,复性后蛋白单体占比约73.60%,与电泳鉴定结果一致,表明经上述变性-复性策略获得的结构稳定、活性增高的重组Neuritin蛋白更接近该蛋白的天然结构。结论 “以单体为主,含有少量的二聚体”的稳定结构,接近重组Neuritin蛋白的天然状态。

Abstract

Objective To investigate the native structure of recombinant Neuritin protein through a denaturation-renaturation strategy and activity assessment. Methods 1)Expression, structural characterization, and activity evaluation of the recombinant protein: The recombinant strain was induced for expression according to the Pichia pastoris protocol, and the protein was purified using hydrophobic chromatography. Immunogenicity was confirmed by Western blot, purity was assessed by reducing SDS-PAGE, and protein structure was analyzed by non-reducing SDS-PAGE and size-exclusion chromatography. Protein activity was evaluated using the CCK8 assay established in our laboratory to measure cell viability. 2)Protein denaturation: Urea and β-mercaptoethanol were selected as denaturants, and the effects of denaturant concentration, denaturation time, and protein concentration on the denaturation of recombinant Neuritin protein were systematically investigated. Denaturation conditions were optimized based on structural analysis and activity testing. 3)Protein renaturation: First, an injection buffer was used to minimize non-specific aggregation between protein molecules; then, a renaturation buffer was applied to allow the protein to spontaneously refold according to the thermodynamically lowest energy state. Renaturation conditions were determined based on structural stability and recovery of biological activity. Results 1)After purification via hydrophobic interaction chromatography, the induced Neuritin protein showed immunological reactivity confirmed by Western blot, with a purity exceeding 95% as determined by reducing SDS-PAGE. Initial structural analysis by non-reducing SDS-PAGE revealed that the recombinant protein was predominantly in dimeric form, with monomers accounting for approximately 31.37%. 2)The effects of Urea and β-mercaptoethanol concentrations, denaturation time, and protein concentration on denaturation were examined. The optimal condition for complete denaturation was determined to be 10 mol·L^-1 Urea and 10 mmol·L^-1 β-mercaptoethanol acting on 0.4 μg·μL^-1 recombinant Neuritin protein for 2 hours. Further size-exclusion chromatography and activity assays confirmed that under these conditions, the protein's spatial structure was fully unfolded and its biological activity was completely lost. 3)Renaturation experiments showed that after 12-36 hours, the protein structure stabilized, predominantly existing as monomers with a small proportion of dimers. Maximum activity was achieved at 24 hours of renaturation. Comparing the renatured protein with the original pre-denatured protein, activity assays indicated enhanced activity in the renatured form. Structural analysis by size-exclusion chromatography revealed that the renatured protein consisted of approximately 73.60% monomers, consistent with electrophoretic results. This indicates that the recombinant Neuritin protein obtained through this denaturation-renaturation strategy exhibits improved structural stability and higher activity, more closely resembling its native conformation. Conclusion A stable structure dominated by monomers with a minor fraction of dimers closely resembles the native state of recombinant Neuritin protein.

关键词

Neuritin / 蛋白天然结构 / 蛋白变性 / 蛋白复性 / 生物学活性鉴定

Key words

Neuritin / native protein structure / protein denaturation / protein renaturation / biological activity characterization

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基金资助

国家重大新药创制项目(2019ZX09301161);国家自然科学基金项目(32060164);新疆生产建设兵团指导性科技计划项目(2025ZD007)