多策略协同提升蛋白质晶体的衍射分辨率

doi:10.13560/j.cnki.biotech.bull.1985.2025-0975

摘要/Abstract

摘要：

目的蛋白质晶体衍射是结构生物学研究的重要方法，晶体的衍射分辨率直接决定了模型的精确度及应用可行性。需要采取多种策略组合优化蛋白质晶体质量，提高晶体的衍射分辨率。方法以催化芳基偶联反应的P450酶为研究对象，利用分子生物学技术构建原核表达系统，并通过镍柱亲和层析和体积排阻色谱等方法进行纯化。在结晶阶段，采用气相扩散法系统筛选了1 632种初始条件，以此为基础，重点对缓冲液pH值、沉淀剂种类与浓度等关键变量进行精细化筛选，并引入多种添加剂以改善结晶环境。进一步结合结构预测分析，对蛋白柔性区域进行理性截短设计，并尝试SUMO标签的融合表达以增强蛋白的稳定性。结果实现了P450蛋白在大肠杆菌中的高效可溶表达，并经两步纯化获得可用于结晶的高纯度蛋白。通过大规模结晶条件筛选与多轮优化，结合序列截短和SUMO标签融合等策略协同作用，晶体形态显著改善，由原来的微晶或无定形沉淀转变为外观规则、棱角清晰的单晶。晶体衍射分辨率由初始的10 Å显著提升至2.86 Å，获得了可用于高分辨率结构解析的优质晶体。结论使用多策略协同的方法成功改善了蛋白晶体形态，提高了蛋白晶体的衍射分辨率。

关键词: 蛋白结晶, X射线衍射, 蛋白纯化, 蛋白表达, 细胞色素P450

Abstract:

Objective Protein crystallographic diffraction is an essential technique in structural biology research. The diffraction resolution of crystals directly determines the accuracy of atomic models and their practical applicability. Therefore, a combination of multiple strategies is required to optimize protein crystallization quality and enhance diffraction resolution. Method The P450 enzyme, which catalyzes aryl coupling reactions, was chosen as the research focus. A prokaryotic expression system was first constructed via molecular biology techniques. The protein was purified using nickel-affinity chromatography and size-exclusion chromatography. During the crystallization stage, 1,632 initial conditions were systematically screened through the vapor diffusion method. Based on preliminary results, key variables including buffer pH, precipitant type and concentration were finely optimized, and various additives were introduced to improve the crystallization environment. Furthermore, rational truncation of flexible regions was designed according to structural predictions, and SUMO tag fusion was attempted to enhance protein stability. Result The P450 protein was successfully expressed solubly at high levels in E. coli and purified to high purity via a two-step purification process, making it suitable for crystallization screening. Through large-scale crystallization condition screening and multiple rounds of optimization, combined with synergistic strategies including sequence truncation and SUMO fusion, crystal morphology was significantly improved—transitioning from initial microcrystals or amorphous precipitates to well-defined single crystals with regular shapes and sharp edges. The diffraction resolution of the crystals was markedly enhanced from an initial 10 Å to 2.86 Å, yielding high-quality crystals suitable for high-resolution structural analysis. Conclusion By employing a multi-strategy synergy, the protein crystal morphology and diffraction resolution of protein crystals is significantly enhanced.

Key words: protein crystallization, X-ray diffraction, protein purification, protein expression, cytochrome P450

任泓宇, 庞翠萍, 古阳, 周佳海. 多策略协同提升蛋白质晶体的衍射分辨率[J]. 生物技术通报, 2026, 42(1): 76-85.

REN Hong-yu, PANG Cui-ping, GU Yang, ZHOU Jia-hai. Multi-strategy Synergy Enhances the Diffraction Resolution of Protein Crystals[J]. Biotechnology Bulletin, 2026, 42(1): 76-85.

图/表 12

图1 P450表达标签替换及分子伴侣共表达A-C：分别为P450KstB、P450MciB、P450ScnB添加分子伴侣及替换融合标签的表达情况，其中编号1为未诱导菌株，编号2为SUMO-P450，编号3-5为SUMO-P450分别添加分子伴侣pGro7、pTF16和pKJE7，编号6为TF-P450，编号7为MBP-P450；D：替换GST标签的表达情况，编号1为未诱导菌株，编号2-4分别为添加GST标签的P450KstB、P450ScnB和P450MciB

Fig. 1 Replacing expression tags and co-expressing molecular chaperones of P450A-C: The expression of P450KstB, P450MciB, and P450ScnB, respectively, with molecular chaperone co-expression and fusion tag swapping. 1: Uninduced strain, 2: SUMO-P450, 3-5: the SUMO-P450 strain was introduced with the molecular chaperones pGro7, pTF16, and pKJE7, respectively, 6: TF-P450, 7: MBP-P450. D: The expression after replacing the GST tag. 1: Uninduced strain, 2-4: P450KstB, P450ScnB, and P450MciB fused with GST tag, respectively

图2 可溶蛋白的Ni柱亲和层析结果分析A-C：分别为使用镍柱对pCold-TF-KstB、pRSF-sumo-KstB+pGro7和pMALc6T-MBP-MciB进行亲和层析的纯化结果

Fig. 2 Ni-NTA analysis of soluble proteins results by Ni-affinity chromatographyA-C: The purification results of affinity chromatography using nickel columns for pCold-TF-KstB, pRSF-sumo-KstB+pGro7, and pMALc6T-MBP-MciB, respectively

图3 P450GpeC蛋白的表达情况分析A：P450GpeC的细胞破碎液及沉淀/上清的SDS-PAGE分析；B：P450GpeC的紫外吸收光谱，425 nm处为特征吸收

Fig. 3 Analysis of P450GpeC protein expressionA: The cell lysate of P450GpeC and the SDS-PAGE analysis of precipitate and supernatant. B: The UV absorption spectrum of P450GpeC with a characteristic absorption peak at 425 nm

图4 P450GpeC的Ni柱亲和层析

Fig. 4 Analysis of P450GpeC results by Ni-affinity chromatography

图5 体积排阻色谱纯化P450GpeC的洗脱色谱图及SDS-PAGE分析结果

Fig. 5 Elution chromatogram and SDS-PAGE analysis results of P450GpeC purified by SEC

图6 AlphaFold 3预测的P450GpeC蛋白结构分析

Fig. 6 Structural analysis of the P450GpeC protein predicted by AlphaFold 3

图7 P450GpeC初筛后不同结晶条件下的晶体形态及衍射图箭头所示为用于衍射的晶体

Fig. 7 Crystal images and diffraction patterns of P450GpeC under various initial crystallization conditionsThe arrow indicates the crystal used for diffraction

图8 添加硫酸铵及晶种法优化后的P450GpeC晶体及衍射图箭头所示为用于衍射的晶体

Fig. 8 Crystal images and diffraction patterns of P450GpeC optimized by ammonium sulfate addition and seeding methodThe arrow indicates the crystal used for diffraction

图9 SUMO-P450GpeC-ΔC8的Ni柱亲和层析

Fig. 9 Analysis of SUMO-P450GpeC-ΔC8 results by Ni-affinity chromatography

图10 体积排阻色谱纯化SUMO-P450GpeC-△C8的洗脱色谱图及SDS-PAGE分析结果

Fig. 10 Elution chromatogram and SDS-PAGE analysis results of SUMO-P450GpeC-△C8 purified by SEC

图11 对SUMO-P450GpeC-△C8优化后的晶体形态及衍射图箭头所示为用于衍射的晶体

Fig. 11 Crystal images and diffraction patterns of optimized SUMO-P450GpeC-△C8 crystalsThe arrow indicates the crystal used for diffraction

表1 晶体优化方式及效果

Table 1 Crystal optimization methods and effects

优化方式 Optimization method	优化效果 Optimization result	分辨率 Resolution （Å）
结晶初筛	无法获得蛋白晶体	-
C端loop截短	晶体过小或者表面粗糙	10
溶剂优化	改善晶体形态	5-8
添加硫酸铵	依附盐晶生长	3-4
添加晶种	脱离盐晶	3-4
添加SUMO标签	外观规则且棱角清晰的高质量集体	2.86

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