Sun Ziyong, Chen Junyong, Yao Hongwei, Liu Lili, Wang Jing, Zhang Jing, Liu Jian-Ning
Institute of Molecular Medicine, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 22 Hankou Road, 210093 Nanjing, China.
Protein Expr Purif. 2005 Sep;43(1):26-32. doi: 10.1016/j.pep.2005.05.005.
To prevent in vivo degradation, small peptides are usually expressed in fusion proteins from which target peptides can be released by proteolytic or chemical reagents. In this report, a modified Ssp dnaB mini-intein linked with a chitin binding domain tag was used as a fusion partner for production of human brain natriuretic peptide (hBNP), a hormone for the treatment of congestive heart failure. The fusion protein was expressed as an inclusion body in Escherichia coli. After refolding, the fusion protein was purified with a chitin affinity column, and dnaB mini-intein mediated peptide-bond hydrolysis was triggered by shifting the pH in the chitin column to 7.0 at 25 degrees C for 16 h, which led to the release and separation of hBNP from its fusion partner. The hBNP sample was further purified with reverse phase HPLC and its biological activity was assayed in vitro. It was found that hBNP had a potent vasodilatory effect on rabbit aortic strips with an EC(50) of (1.24+/-0.32)x10(-6)mg/ml, which was similar to that of the synthetic BNP standard. The expression strategy described here promises to produce small peptides without use of proteolytic or chemical reagents.
为防止体内降解，小肽通常在融合蛋白中表达，通过蛋白水解或化学试剂可从中释放出目标肽。在本报告中，一种与几丁质结合域标签相连的修饰型Ssp dnaB微小内含肽被用作融合伙伴，用于生产人脑钠肽（hBNP），一种用于治疗充血性心力衰竭的激素。该融合蛋白在大肠杆菌中以包涵体形式表达。复性后，融合蛋白用几丁质亲和柱纯化，通过在25℃将几丁质柱中的pH值调至7.0并保持16小时，触发dnaB微小内含肽介导的肽键水解，从而使hBNP从其融合伙伴中释放并分离出来。hBNP样品再用反相高效液相色谱进一步纯化，并对其体外生物活性进行测定。结果发现，hBNP对兔主动脉条具有强大的舒张血管作用，其半数有效浓度（EC50）为（1.24±0.32）×10-6mg/ml，与合成的BNP标准品相似。本文所述的表达策略有望在不使用蛋白水解或化学试剂的情况下生产小肽。
