Zhou Ying, Ma Xue, Hou Zheng, Xue Xiaoyan, Meng Jingru, Li Mingkai, Jia Min, Luo Xiaoxing
Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
Protein Expr Purif. 2012 Sep;85(1):38-43. doi: 10.1016/j.pep.2012.06.016. Epub 2012 Jul 6.
Glucagon-like peptide-1 (GLP-1)(2) has been attracting increasing interest on account of its prominent benefits in type 2 diabetes. However, its clinical applications are limited by the short half-life in vivo. To overcome this limitation, a new polymer of GLP-1 was developed by prodrug strategy. In this study a recombinant protein, rhGLPs, was successfully constructed, cloned into plasmid pET30a (+) and expressed in Escherichia coli ArcticExpress(DE3)RP in the form of inclusion body. The recombinant fusion protein productivity could be enhanced by high cell density culture of the recombinant strain. As a result, about 40 g wet weight cells per liter were obtained. The protein was purified by size-exclusion chromatography on a Superdex 75 column and refolded using reverse dilution and dialysis methods. SDS-PAGE, HPLC and MALDI-TOF mass spectrometry were undertaken to determine the purity and molecular weight of rhGLPs. Bioactivity assay revealed that it had glucose-lowering and insulin-releasing action in vivo.
胰高血糖素样肽-1（GLP-1）(2)因其在2型糖尿病中的显著益处而受到越来越多的关注。然而，其临床应用受到体内半衰期短的限制。为克服这一限制，通过前药策略开发了一种新的GLP-1聚合物。在本研究中，成功构建了一种重组蛋白rhGLPs，将其克隆到质粒pET30a(+)中，并以包涵体形式在大肠杆菌ArcticExpress(DE3)RP中表达。通过重组菌株的高细胞密度培养可以提高重组融合蛋白的产量。结果，每升获得约40 g湿重细胞。该蛋白通过Superdex 75柱上的尺寸排阻色谱法纯化，并使用反向稀释和透析方法复性。采用SDS-PAGE、HPLC和MALDI-TOF质谱法测定rhGLPs的纯度和分子量。生物活性测定表明，它在体内具有降血糖和促胰岛素释放作用。
