Sun Ziyong, Lu Wei, Tang Yanchun, Zhang Jing, Chen Junyong, Deng Hualing, Li Xuerong, Liu Jian-Ning
Institute of Molecular Medicine and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 22 Hankou Road, Nanjing 210093, China.
Protein Expr Purif. 2007 Oct;55(2):312-8. doi: 10.1016/j.pep.2007.04.019. Epub 2007 May 3.
Urodilatin is a 32-amino acid peptide hormone synthesized in kidney to regulate natriuresis and diuresis. It has been shown clinically useful for the treatment of acute decompensated heart failure. A synthetic deoxyoligonucleotide encoding urodilatin was cloned into a pET32a vector immediately after the thioredoxin encoding sequence with a hexa-hisditine tag and an enterokinase recognition site incorporated in between. The fusion protein was overexpressed in Escherichia coli, which constituted 28% of the total cell proteins. More than 85% of Trx-urodilatin was soluble and purified nearly homogenous by Ni-Sepharose affinity chromatography. Urodilatin was then released from the fusion protein by the enterokinase treatment and separated from the fusion partner by the subtractive chromatography using Ni-Sepharose once again. The urodilatin sample was further purified with reverse phase HPLC. Via a biological activity assayed in vitro, it was found that urodilatin had a potent vasodilatory effect on rabbit aortic strips with an EC50 of (2.02+/-0.36)x10(-6)mg/ml, which was similar to that of the synthetic urodilatin standard. The method described here promises to produce about 4.5mg fully active recombinant urodilatin with homogeneity over 97% from one liter shaking flask culture of E. coli.
尿舒张素是一种在肾脏中合成的由32个氨基酸组成的肽类激素，用于调节尿钠排泄和利尿。临床研究表明，它对治疗急性失代偿性心力衰竭有效。将编码尿舒张素的合成脱氧寡核苷酸克隆到pET32a载体中，紧接在硫氧还蛋白编码序列之后，中间插入一个六组氨酸标签和一个肠激酶识别位点。融合蛋白在大肠杆菌中过量表达，占总细胞蛋白的28%。超过85%的Trx-尿舒张素是可溶的，通过镍琼脂糖亲和层析纯化得到几乎均一的产物。然后通过肠激酶处理从融合蛋白中释放出尿舒张素，并再次使用镍琼脂糖通过减法层析将其与融合伙伴分离。尿舒张素样品通过反相高效液相色谱进一步纯化。通过体外生物活性测定发现，尿舒张素对兔主动脉条具有强大的血管舒张作用，EC50为(2.02±0.36)×10(-6)mg/ml，与合成尿舒张素标准品相似。本文所述方法有望从一升大肠杆菌摇瓶培养物中产生约4.5mg活性完全且纯度超过97%的重组尿舒张素。
