High-Level Soluble Expression of Recombinant Human Bone Morphogenetic Protein-2 in Escherichia coli

IF 3.9 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2025-09-09 DOI:10.1021/acssynbio.5c00392

Youming Cui, Yanqiu Liang, Bin Zhang, Quanfeng Liang, Yi Wang and Wei Luo*,

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Abstract

Human Bone Morphogenetic Protein-2 (hBMP-2) serves as a critical regulator in bone and cartilage formation; however, its industrial application is hindered by its inherent tendency to form inclusion bodies in prokaryotic expression systems. To address this issue, we established a recombinant hBMP-2 (rhBMP-2) expression system using the pCold II plasmid and the SHuffle T7 strain. We explored several strategies to enhance the solubility of rhBMP-2, including coexpression with molecular chaperones, vesicle-mediated secretory expression, fusion expression with synthetic intrinsically disordered proteins (SynIDPs), and fusion expression with small-molecule peptide tags. Our results showed that coexpression with the molecular chaperone pGro7 significantly improved the solubility of rhBMP-2. Fusion with SynIDPs led to complete solubility of rhBMP-2; however, the protein was expressed exclusively in the monomeric form. Among the tested small-molecule peptide tags, GB1 was the most effective, achieving fully soluble rhBMP-2 expression. Western blot analysis confirmed the coexistence of monomeric and dimeric forms of rhBMP-2. Subsequent purification of rhBMP-2 through metal chelate chromatography resulted in an expression level of 109.7 ± 5.0 mg·L^–1. In summary, we successfully demonstrated fully soluble expression of rhBMP-2 in Escherichia coli, providing a valuable foundation for its industrial-scale production.

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重组人骨形态发生蛋白-2在大肠杆菌中的高可溶性表达。

人骨形态发生蛋白-2 （hBMP-2）在骨和软骨形成中起关键调节作用；然而，其在原核表达系统中形成包涵体的固有倾向阻碍了其工业应用。为了解决这一问题，我们利用pCold II质粒和SHuffle T7菌株建立了重组hBMP-2 （rhBMP-2）表达系统。我们探索了几种提高rhBMP-2溶解度的策略，包括与分子伴侣共表达、囊泡介导的分泌表达、与合成内在无序蛋白（SynIDPs）融合表达以及与小分子肽标签融合表达。结果表明，与分子伴侣pGro7共表达可显著提高rhBMP-2的溶解度。与SynIDPs融合导致rhBMP-2完全溶解；然而，该蛋白仅以单体形式表达。在测试的小分子肽标签中，GB1是最有效的，实现了rhBMP-2的完全可溶性表达。Western blot分析证实了rhBMP-2的单体和二聚体共存。随后通过金属螯合层析纯化rhBMP-2，表达量为109.7±5.0 mg·L-1。总之，我们成功地证明了rhBMP-2在大肠杆菌中完全可溶表达，为其工业规模生产提供了有价值的基础。

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来源期刊

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

期刊介绍： The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.