Ding Yuedi, Peng Ying, Deng Lili, Wu Yu, Fu Qiang, Jin Jian
Lab. Drug Design & Molecular Pharmcology, Department of Pharmaceutical Engineering, School of Pharmaceutical Sciences, Jiangnan University, 1800 Lihu Rd, Wuxi, Jiangsu 214122, China.
Curr Pharm Biotechnol. 2014;15(9):856-63. doi: 10.2174/1389201015666141012182106.
Human brain natriuretic peptide (BNP) is utilized in the treatment of acute decompensated congestive heart failure. However, BNP has limited clinical use owing to its rapid clearance and the need for continuous intravenous infusion. Thus, we generated human serum albumin (HSA)-BNP fusion constructs to produce long-acting fusion proteins in Pichia pastoris. Four fusion proteins, BNP-HSA, (BNP)2-HSA, (BNP)4-HSA, and HSA-(BNP)2, were constructed, with different numbers of BNP molecules and fusion orientations. BNP-HSA was most abundantly expressed in Pichia pastoris and (BNP)4-HSA had the lowest yield, probably because of the high copy number of BNP. Western blot analysis confirmed the immunogenicity of both BNP and HSA for the four fusion proteins. A cGMP activity assay was used to measure the ability of fusion proteins to stimulate intracellular cGMP expression. Results showed that the fusion protein HSA-(BNP)2 activated human natriuretic peptide receptor A (hNPR-A) with potency similar to that of BNP, despite using a 10-fold higher dosage than BNP. The other three fusion proteins (BNP-HSA, (BNP)2-HSA, and (BNP)4-HSA), only slightly increased NPR-A activity. In addition, fusion with HSA successfully prolonged BNP bioactivity, stimulating intracellular cGMP expression over 24 h. In conclusion, HSA-(BNP)2, with two BNP molecules fused at the C-terminus of HSA, has the highest and most prolonged BNP bioactivity in activating human NPR-A.
人脑利钠肽（BNP）用于治疗急性失代偿性充血性心力衰竭。然而，由于BNP清除迅速且需要持续静脉输注，其临床应用受到限制。因此，我们构建了人血清白蛋白（HSA）-BNP融合构建体，以在毕赤酵母中产生长效融合蛋白。构建了四种融合蛋白，即BNP-HSA、(BNP)2-HSA、(BNP)4-HSA和HSA-(BNP)2，它们具有不同数量的BNP分子和融合方向。BNP-HSA在毕赤酵母中表达量最高，而(BNP)4-HSA产量最低，这可能是由于BNP的高拷贝数所致。蛋白质免疫印迹分析证实了这四种融合蛋白中BNP和HSA的免疫原性。采用环磷酸鸟苷（cGMP）活性测定法来检测融合蛋白刺激细胞内cGMP表达的能力。结果表明，融合蛋白HSA-(BNP)2激活人利钠肽受体A（hNPR-A）的效力与BNP相似，尽管其用量比BNP高10倍。其他三种融合蛋白（BNP-HSA、(BNP)2-HSA和(BNP)4-HSA）仅略微增加了NPR-A活性。此外，与HSA融合成功延长了BNP的生物活性，在24小时以上刺激细胞内cGMP表达。总之，在HSA的C末端融合两个BNP分子的HSA-(BNP)2在激活人NPR-A方面具有最高且最持久的BNP生物活性。
