Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Chines Ministry of Education, Jinan University, Guangzhou 510632, China.
Protein Pept Lett. 2021;28(5):533-542. doi: 10.2174/0929866527666201110123426.
Human growth hormone (hGH) is the first recombinant protein approved for the treatment of human growth hormone deficiency. However, expression in inclusion bodies and low expression levels are enormous challenges for heterologous expression of hGH in Escherichia coli.
To increase the soluble expression of recombinant hGH with correct folding in E. coli.
We constructed a new recombinant expression plasmid containing the coding sequence of the outer membrane protein A (ompA3) which was used for the expression in Transetta (DE3) E. coli. In order to simplify the purification process and cleavage of recombinant proteins, the fusion sequence should contain hexahistidine-tag (His) and enterokinase recognition sites (DK). The effect of different expression conditions on recombinant hGH expression was optimized in flask cultivations. Furthermore, the periplasmic solution containing soluble hGH was purified by Ni-NTA affinity chromatography. Circular dichroism (CD), western blot and mass spectrometry analyses were used to characterize the protein. Moreover, the growth-promoting effect of the purified hGH was also evaluated by cell proliferation assay.
High-level expression (800 μg/mL) was achieved by induction with 0.5 mM IPTG at 30°C for 10 hours. The purity of hGH was over 90%. The immunological activity, secondary structure and molecular weight of the purified hGH were consistent with native hGH. The purified hGH was found to promote the growth of MC3T3-E1 cells, and was found to show the highest activity at a concentration of 100 ng/mL.
Our research provides a feasible and convenient method for the soluble expression of recombinant hGH in E. coli, and may lay a foundation for the production and application of hGH in the industry.
人生长激素（hGH）是第一个获准用于治疗人类生长激素缺乏症的重组蛋白。然而，在大肠杆菌中异源表达 hGH 时，包涵体的形成和低表达水平是巨大的挑战。
提高重组 hGH 在大肠杆菌中的可溶性表达和正确折叠。
我们构建了一个新的重组表达质粒，其中包含外膜蛋白 A（ompA3）的编码序列，用于 Transetta（DE3）大肠杆菌的表达。为了简化重组蛋白的纯化过程和切割，融合序列应含有六组氨酸标签（His）和肠激酶识别位点（DK）。在摇瓶培养中优化了不同表达条件对重组 hGH 表达的影响。此外，通过 Ni-NTA 亲和层析纯化含有可溶性 hGH 的周质溶液。采用圆二色性（CD）、western blot 和质谱分析对蛋白进行表征。此外，还通过细胞增殖试验评估了纯化的 hGH 的促生长作用。
通过在 30°C 下用 0.5mM IPTG 诱导 10 小时，实现了 800μg/mL 的高水平表达。hGH 的纯度超过 90%。纯化的 hGH 的免疫活性、二级结构和分子量与天然 hGH 一致。纯化的 hGH 被发现能促进 MC3T3-E1 细胞的生长，在浓度为 100ng/mL 时表现出最高的活性。
我们的研究为大肠杆菌中重组 hGH 的可溶性表达提供了一种可行且方便的方法，可能为 hGH 的生产和工业应用奠定基础。
