Production of functional human nerve growth factor from the submandibular glands of mice using a CRISPR/Cas9 genome edit
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(2020) 36:176
ORIGINAL PAPER
Production of functional human nerve growth factor from the submandibular glands of mice using a CRISPR/Cas9 genome editing system Yi Gu1,3 · Hui Cao2 · Fei Li2 · Jianli Yu1,3 · Rui Nian1 · Dongxiao Feng2 · Jingtao Lin4 · Haipeng Song2 · Wenshuai Liu1 Received: 15 June 2020 / Accepted: 14 October 2020 © Springer Nature B.V. 2020
Abstract Nerve growth factor (NGF) is an essential trophic factor for the growth and survival of neurons in the central and peripheral nervous systems. For many years, mouse NGF (mNGF) has been used to treat various neuronal and non-neuronal disorders. However, the biological activity of human NGF (hNGF) is significantly higher than that of mNGF in human cells. Using the CRISPR/Cas9 system, we constructed the transgenic mice expressing hNGF specifically in their submandibular glands. As demonstrated by fluorescence immunohistochemical staining, these mice produced hNGF successfully, with 0.8 mg produced per gram of submandibular glands. hNGF with 99% purity was successfully extracted by two-step ion-exchange chromatography and one-step size-exclusion chromatography from the submandibular glands of these transgenic mice. Further, the purified hNGF was verified by LC–MS/MS. We analyzed the NH2-terminus of hNGF using both Edman degradation and LC–MS/MS-based methods. Both results showed that the obtained hNGF lost the NH2-terminal octapeptide (SSSHPIFH). Moreover, the produced hNGF demonstrated a strong promotion in the proliferation of TF1 cells. Keywords CRISPR/Cas9 · Mice · NGF · Submandibular glands · Transgenic
Introduction
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11274-020-02951-x) contains supplementary material, which is available to authorized users. * Haipeng Song [email protected] * Wenshuai Liu [email protected] 1
CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China
2
Shenzhen Innova Nanobodi Co., Ltd, No. 7018 Caitian Road, Shenzhen 518000, China
3
University of Chinese Academy of Sciences, No. 19(A) Yuquan Road, Beijing 100049, China
4
Dalang Hospital of Dongguan, No. 85 Jinlangzhong Road, Dalang 523770, Dongguan, China
Nerve growth factor (NGF) is the prototype of the neurotrophin family, discovered in 1950 (Levimontalcini 1987). It is an essential trophic factor for the survival, growth, and maintenance of neurons in the central and peripheral nervous systems (Ebendal 1989; Hartikka and Hefti 1988). In addition, several non-neuronal cells, including immune cells, brain cells, keratinocytes, blood vessel endothelial cells, etc., have also been proved to be targets of NGF (Aloe and LeviMontalcini 1977; Bischoff and Dahinden 1992; Di Marco et al. 1992; Levimontalcini 1987; Raychaudhuri et al. 2001). Therefore, NGF has been developed as an important therapeutic protein that is used to treat some neurological diseases such as glaucoma and Alzhe
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