蓝藻丝氨酸蛋白酶抑制剂arthropin的制备及其靶蛋白酶的筛选

doi:10.3969/j.issn.1674-8115.2023.04.004

摘要/Abstract

摘要：

目的·制备高纯度的蓝藻丝氨酸蛋白酶抑制剂（serine protease inhibitor，serpin），筛选其靶蛋白酶，并探究其抑制活性。方法·通过氨基酸序列分析，在人类口腔微生物组数据库（eHOMD）发现了一种钝顶节旋藻（Arthrospira platensis）的serpin，将其命名为arthropin；构建融合表达载体pSUMO3-arthropin，将其转入大肠埃希菌BL21（DE3）诱导表达；采用镍离子亲和层析、酶切、反向镍离子亲和层析、阴离子交换层析的4步层析纯化方案将arthropin重组蛋白分离纯化；将arthropin重组蛋白分别与活化的凝血因子Ⅸ（activated factor Ⅸ，FⅨa）、FⅩa、FⅪa、活化的蛋白C（activated protein C，APC）、激肽释放酶1（kallikrein 1，KLK1）等14种丝氨酸蛋白酶共孵育，再利用SDS-PAGE分析共价复合物形成情况，利用反应动力学方法检测arthropin对KLK1的抑制速率。对arthropin重组蛋白的结晶条件进行筛选，选取合适的晶体进行X射线衍射并采集数据。利用AlphaFlod Colab预测arthropin亚稳态结构模型。结果·SDS-PAGE分析发现，arthropin融合蛋白在大肠埃希菌BL21（DE3）中表达；纯化后，获得了高纯度的与理论相对分子质量（45 800）相近的arthropin重组蛋白。将arthropin重组蛋白与14种丝氨酸蛋白酶共孵育后发现，arthropin能与FⅩa、APC、FⅨa、FⅪa、胰蛋白酶（trypsin）、组织蛋白酶G（cathepsin G）、KLK1、KLK7和凝血酶（thrombin）9种蛋白酶形成稳定的共价复合物。Arthropin抑制KLK1的二级反应抑制速率常数为1.7×10³ L/（mol·s）。Arthropin重组蛋白可以在25%聚乙二醇单甲醚550（PEG MME 550）、0.1 mol/L吗啉乙磺酸（MES，pH 6.5）、0.01 mol/L ZnCl₂条件下形成片状晶体，X射线衍射分辨率为10 ?（1 ?=1×10^-10 m）。AlphaFlod Colab预测结果发现，arthropin具有经典的抑制型serpin的结构特点。结论·成功获得高纯度的钝顶节旋藻丝氨酸蛋白酶抑制剂arthropin，其具有较广谱的丝氨酸蛋白酶抑制能力，但抑制速率较低。

关键词: 丝氨酸蛋白酶抑制剂, 钝顶节旋藻, 原核表达, 蛋白结晶

Abstract:

Objective ·To prepare a high-purity cyanobacterial serine protease inhibitor, screen its target proteases, and detect its inhibitory activity. Methods ·A novel serine protease inhibitor from Arthrospira platensis was identified in the Expanded Human Oral Microbiome Database (eHOMD) by amino acid sequence alignment and named as arthropin. The fusion expression vector pSUMO3-arthropin was constructed and transferred into Escherichia coli (E. coli) BL21(DE3) system for fusion protein expression. The recombinant arthropin was purified by a four-step chromatographic purification approach of nickel affinity chromatography, enzymatic digestion, reverse nickel affinity chromatography, and anion exchange chromatography. In addition, the recombinant arthropin was co-incubated with 14 serine proteases such as activated factor Ⅸ (FⅨa), FⅩa, FⅪa, activated protein C (APC) and kallikrein 1 (KLK1), respectively, and then analyzed by SDS-PAGE. The inhibitory rate of arthropin on KLK1 was assayed with kinetic methods. The crystallization conditions of the recombinant arthropin were screened preliminarily, and the suitable crystals were picked for X-ray diffraction to collect the data. Finally, a sub-stable structure model of arthropin was predicted with AlphaFlod Colab. Results ·SDS-PAGE showed that the fused arthropin was successfully expressed in the E. coli BL21(DE3) system, and following purification, the high-purity recombinant arthropin, the relative molecular mass of which was similar to the theoretical value (45 800), was obtained. The co-incubation analysis of recombinant arthropin with 14 serine proteases revealed that arthropin was able to form stable covalent complexes with 9 proteases, including FⅩa, APC, FⅨa, FⅪa, trypsin, cathepsin G, KLK1, KLK7 and thrombin. Arthropin inhibited KLK1 with a second-order association rate constant of 1.7×10³ L/(mol·s). Moreover, the recombinant arthropin crystalised under the condition of 25% PEG MME 550, 0.1 mol/L MES (pH 6.5) and 0.01 mol/L ZnCl₂, and the crystals preliminarily diffracted to a resolution of 10 ? (1 ?=1×10^-10 m). The analysis of the structure predicted by AlphaFlod Colab revealed that arthropin had the classical structural features of the inhibitory serpin. Conclusion ·Arthropin, a serpin from Arthrospira platensis, was successfully obtained with high purity and a broad-spectrum of serine protease inhibition, but at a low inhibitory rate.

Key words: serine protease inhibitor (serpin), Arthrospira platensis, prokaryotic expression, protein crystallization

中图分类号:

许佳伟, 周爱武, 杨愈丰. 蓝藻丝氨酸蛋白酶抑制剂arthropin的制备及其靶蛋白酶的筛选[J]. 上海交通大学学报（医学版）, 2023, 43(4): 428-436.

XU Jiawei, ZHOU Aiwu, YANG Yufeng. Preparation and target protease identification of a cyanobacterial serine protease inhibitor, arthropin[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(4): 428-436.

图/表 5

图1 Arthropin与多种serpin的RCL序列比对Note：α1-AT—α1-antitrypsin (Uniprot ID: A0A024R6I7); KAL—kallistatin (Uniprot ID: P29622); PN1—protein nexin 1 (Uniprot ID: P07093); miropin (Uniprot ID: G8UQY8); siropin (UniProt ID: R6RSK2); arthropin (GenBank ID: AMW27298.1). s1C—strand 1 of β-sheet C.

Fig 1 RCL sequence comparison of arthropin with various serpins

图2 SDS-PAGE分析arthropin融合蛋白的表达、酶切与纯化Note：A. Protein expression in the whole bacteria and lysis supernatant of E. coli before and after induction. Lane 1—marker; lane 2—total bacterial protein sample before induction; lane 3—supernatant protein sample before induction; lane 4—total bacterial protein sample after induction for 12 h; lane 5—supernatant protein sample after induction for 12 h. B. Enzymatic digestion and purification by nickel ion affinity chromatography. Lane 1—marker; lane 2—protein sample before SENP2 digestion; lane 3—protein sample after SENP2 digestion; lane 4?5—flow-through solution of the nickel column after SENP2 digestion; lane 6?7—washing solution of the nickel column after SENP2 digestion; lane 8—elution solution of the nickel column after SENP2 digestion. C. Purification by anion-exchange chromatography. Lane 1—marker; lane 2?12—protein samples eluted by a continuous gradient of 145?685 mmol/L NaCl. The black arrow indicates the fusion protein and the red arrows indicate the monomeric protein.

Fig 2 Expression, enzymatic digestion and purification of arthropin fusion protein analyzed by SDS-PAGE

图3 Arthropin靶蛋白酶的筛选与反应动力学检测Note：A. Arthropin (3 μg) was incubated with the proteases (0.4 μg), respectively, and the complex formation was analyzed by SDS-PAGE. The red arrows indicate the complexes of arthropin with the target proteases. M—marker; Try—trypsin; Arth—arthropin; Cpx—arthropin-proteinase covalent complex. B. Determination of the SI (left panel) and the kapp (right panel) of arthropin's inhibition of KLK1.

Fig 3 Screening and reaction kinetics assays for target proteases of arthropin

图4 不同结晶条件筛选得到的arthropin蛋白质晶体(×6.3)Note：A. 25% PEG MME 550, 0.1 mol/L MES (pH 6.5), and 0.01 mol/L ZnCl2. B. 20% PEG 4000, 20% iso-propanol, and 0.1 mol/L sodium citrate (pH 5.6). C. 0.4 mol/L NaH2PO4, 1.6 mol/L K2HPO4, 0.1 mol/L imidazole (pH 8.0), and 0.2 mol/L NaCl (final pH 7.6).

Fig 4 Arthropin crystals from different crystallization conditions (×6.3)

图5 Arthropin的结构分析Note：A. The arthropin model created with AlphaFlod Colab. β-sheets A, B, and C are indicated in red, blue, and yellow, respectively. RCL is in purple. B. Surface charge diagram of arthropin. The red colour indicates the negative charge and the blue colour indicates the positive charge.

Fig 5 Structural analysis of arthropin

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