表皮生长因子(EGF)蛋白是参与调节细胞生长的关键生物分子,扩散,迁移和分化,用于各种治疗应用,如伤口愈合和组织再生。重组EGF的生产对于研究其生物学功能和临床翻译至关重要。然而,在原核细胞中表达的EGF蛋白通常发生在包涵体中,与可溶性标记蛋白共表达是制备重组EGF的有效方法。在这项研究中,我们表达了与HaloTag(Halo-rhEGF)融合的重组人EGF(rhEGF),并且在可溶性部分中发现了大部分Halo-rhEGF。细胞生长实验表明纯化的Halo-rhEGF蛋白能促进成纤维细胞(NIH3T3)和上皮细胞(HaCaT)的增殖,并显著提高了他们的生存能力。细胞内信号蛋白的磷酸化,ERK1/2和c-Jun,通过Halo-rhEGF处理刺激,调节细胞增殖的蛋白质的表达水平显着增加。RNA测序分析表明,rhEGF可以增加富含核糖体生成和细胞增殖的基因的转录。此外,Halo-rhEGF可以被HaloTag配体标记用于荧光成像,并且可以通过与阴离子生物材料结合在组织修复中缓慢释放。总之,HaloTag是一种高效的rhEGF蛋白表达融合标签,纯化和控释,Halo-rhEGF可以促进上皮细胞和成纤维细胞的增殖和活力。
Epidermal growth factor (EGF) protein is a crucial biomolecule involved in regulating cell growth, proliferation, migration and differentiation, which is used in various therapeutic applications, such as wound healing and tissue regeneration. The production of recombinant EGF is essential for studying its biological function and for its clinical translation. However, EGF protein expressed in prokaryotic cells often occurs in inclusion bodies, and co-expression with soluble tag protein is an effective method to prepare recombinant EGF. In this study, we expressed recombinant human EGF (rhEGF) fused to a HaloTag (Halo-rhEGF) and a large portion of Halo-rhEGF was found in the soluble fraction. Cell growth assay showed that the purified Halo-rhEGF protein could promote the proliferation of fibroblasts (NIH 3T3) and epithelial cells (HaCaT), and significantly increased their viability. Phosphorylation of the intracellular signaling proteins, ERK1/2 and c-Jun, was stimulated by treatment with Halo-rhEGF and the expression levels of proteins regulating cell proliferation were significantly increased. RNA sequencing analysis revealed that rhEGF could increase the transcription of genes enriched in ribosome generation and cell proliferation. Moreover, Halo-rhEGF can be labelled by HaloTag ligand for fluorescence imaging and can be slowly released in tissue repair by binding to anion biomaterials. In conclusion, HaloTag is an efficient fusion tag for rhEGF protein expression, purification and controlled release, and Halo-rhEGF can promote the proliferation and viability of epithelial and fibroblast cells.