Shamriz Shabnam, Ofoghi Hamideh, Amini-Bayat Zahra
Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran, Iran.
Iran J Biotechnol. 2016 Mar;14(1):16-22. doi: 10.15171/ijb.1331.
Pivotal roles of Nerve growth factor (NGF) in the development and survival of both neuronal and non-neuronal cells indicate its potential for the treatment of neurodegenerative diseases. However, investigation of NGF deficits in different diseases requires the availability of properly folded human β-NGF. In previous studies bacterial expression of hNGF demonstrated the feasibility of its overproduction. However, known limitations in the use of as an expression host for a protein with three intra-chain disulfide bonds were evident.
Here an optimized system was developed to overexpress the soluble NGF in
The gene encoding the β subunit of mature hNGF was optimized based on codon preference and cloned into pET-32a expression vector providing His- and Trx- tags for detection and increasing the solubility of recombinant protein, respectively. The recombinant DNA was expressed in Origami (DE3), which enhances the correct formation of disulfide bonds in the cytoplasm of . Different culture conditions were evaluated to increase soluble expression of the target protein.
The highest soluble expression level was achieved when Origami (DE3) cells expressing NGF were grown at 30ºC in TB medium with 0.2 mM IPTG induction at OD = 1 for 4 h.
Our results indicated that the recombinant NGF was successfully expressed as a soluble form.
神经生长因子（NGF）在神经元和非神经元细胞的发育与存活中发挥关键作用，这表明其在治疗神经退行性疾病方面具有潜力。然而，研究不同疾病中的NGF缺陷需要获得正确折叠的人β-NGF。在先前的研究中，hNGF的细菌表达证明了其过量生产的可行性。然而，作为具有三个链内二硫键的蛋白质的表达宿主，其已知的局限性是明显的。
在此开发了一种优化系统，以在……中过表达可溶性NGF。
基于……密码子偏好对成熟hNGF的β亚基编码基因进行优化，并克隆到pET-32a表达载体中，分别提供His-和Trx-标签用于检测和增加重组蛋白的溶解度。重组DNA在Origami（DE3）中表达，这增强了……细胞质中二硫键的正确形成。评估了不同的培养条件以增加目标蛋白的可溶性表达。
当表达NGF的Origami（DE3）细胞在30℃的TB培养基中，在OD = 1时用0.2 mM IPTG诱导培养4小时时，可实现最高的可溶性表达水平。
我们的结果表明重组NGF成功表达为可溶性形式。
