Journal of Shanghai Jiao Tong University (Medical Science) >

2023 , Vol. 43 >Issue 4: 428 - 436

DOI: https://doi.org/10.3969/j.issn.1674-8115.2023.04.004

Basic research

Preparation and target protease identification of a cyanobacterial serine protease inhibitor, arthropin

  • Jiawei XU ,
  • Aiwu ZHOU ,
  • Yufeng YANG
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  • 1.Department of Biological Engineering, Zhuhai Campus of Zunyi Medical University, Zhuhai 519040, China
    2.Department of Pathophysiology, College of Basic Medical Sciences, Shanghai Jiao Tong University, Shanghai 200025, China
YANG Yufeng, E-mail: yfyang@zmc.edu.cn.

Received date: 2022-12-08

  Accepted date: 2023-04-03

  Online published: 2023-04-28

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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

Cite this article

Jiawei XU , Aiwu ZHOU , Yufeng YANG . 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 . DOI: 10.3969/j.issn.1674-8115.2023.04.004

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