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Transdermal peptide conjugated to human connective tissue growth factor with enhanced cell proliferation and hyaluronic acid synthesis activities produced by a silkworm silk gland bioreactor
Applied Microbiology and Biotechnology ( IF 5 ) Pub Date : 2020-10-19 , DOI: 10.1007/s00253-020-10836-0
Yuancheng Wang 1, 2 , Feng Wang 1, 2, 3 , Sheng Xu 1, 2 , Riyuan Wang 1, 2 , Chi Tian 1, 2 , Yanting Ji 1, 2 , Qianqian Yang 1, 2 , Ping Zhao 1, 2 , Qingyou Xia 1, 2, 3
Affiliation  

Abstract

Human connective tissue growth factor (CTGF) is a secreted cysteine-rich peptide that stimulates cell proliferation, migration, and extracellular matrix production during tissue development, differentiation, angiogenesis, implantation, wound healing, and fibrosis processes, with broad application in the medical and cosmetic medical fields. However, the production of CTGF is currently limited by its low yield and purity in current bioreactors. In this study, two genetically modified silkworm strains were generated harboring artificially designed CTGF-8ht and pepCTGF-8ht genes, respectively, that contain an enhanced His-tag with eight histidine residues with or without a transdermal peptide (pep). Both recombinant CTGF-8ht and pepCTGF-8ht proteins were successfully expressed in the silkworm silk gland and cocoon, and could be easily extracted and purified from the cocoon by single-affinity immunoprecipitation column chromatography, achieving a purity of more than 95%. Moreover, compared with CTGF-8ht protein, pepCTGF-8ht protein exhibited better cell proliferation activity by activating the extracellular signal-regulated kinase (ERK) pathway and enhanced hyaluronic acid synthesis activity by upregulating hyaluronan synthase 3 expression; moreover, the addition of pep significantly improved the transmembrane ability of CTGF-8ht protein. These results should help to promote the application prospects of CTGF and further guide the design and development of protein drugs from silkworm and other bioreactor systems.

Key points

  • A silkworm bioreactor was optimized to produce connective tissue growth factor (CTGF)

  • The transgene contained an enhanced 8-His-tag and transmembrane peptide (pep)

  • Recombinant CTGF was easily purified with maintained or higher biological activity



中文翻译:

由蚕丝腺生物反应器产生的与人结缔组织生长因子结合的透皮肽具有增强的细胞增殖和透明质酸合成活性

摘要

人结缔组织生长因子 (CTGF) 是一种分泌的富含半胱氨酸的肽,可在组织发育、分化、血管生成、植入、伤口愈合和纤维化过程中刺激细胞增殖、迁移和细胞外基质产生,在医学和美容医学领域。然而,目前 CTGF 的生产受限于其在当前生物反应器中的低产率和纯度。在这项研究中,产生了两种含有人工设计的 CTGF-8htpepCTGF-8ht的转基因家蚕品系。基因,分别含有增强的组氨酸标签,带有或不带有透皮肽(pep)的八个组氨酸残基。重组CTGF-8ht和pepCTGF-8ht蛋白均成功地在蚕丝腺和蚕茧中表达,通过单亲和免疫沉淀柱层析可以很容易地从蚕茧中提取纯化,纯度达到95%以上。此外,与CTGF-8ht蛋白相比,pepCTGF-8ht蛋白通过激活细胞外信号调节激酶(ERK)通路表现出更好的细胞增殖活性,并通过上调透明质酸合酶3的表达增强透明质酸合成活性;此外,pep的加入显着提高了CTGF-8ht蛋白的跨膜能力。

关键点

  • 优化家蚕生物反应器以生产结缔组织生长因子 (CTGF)

  • 转基因含有增强的 8-His 标签和跨膜肽 (pep)

  • 重组 CTGF 易于纯化,具有保持或更高的生物活性

更新日期:2020-11-15
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