The Effect of Albumin Fusion Patterns on the Production and Bioactivity of the Somatostatin-14 Fusion Protein in Pichia pastoris
The Effect of Albumin Fusion Patterns on the Production and Bioactivity of the Somatostatin-14 Fusion Protein in Pichia pastoris
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DOI：
10.1007/s12010-013-0304-1
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发表时间：
2013-08-01
影响因子：
3
通讯作者：
Fu, Qiang
中科院分区：
文献类型：
--
作者：
Ding, Yuedi;Fan, Jun;Fu, Qiang
生长抑素是生长激素的天然抑制剂，其类似物在临床上用于治疗肢端肥大症、肥大症、促甲状腺激素瘤和其他类癌综合征。然而，天然生长抑素由于其体内半衰期短而限制了其临床应用。利用白蛋白融合技术构建长效融合蛋白，并以毕赤酵母为表达系统。以生长抑素14（SS）14的不同融合拷贝和融合方向构建了（SS 14）（2）-人血清白蛋白（HSA）、（SS 14）（3）-HSA和HSA-（SS 14）（3）三种融合蛋白。由于生长抑素-14分子的额外融合，（SS 14）（3）-HSA和HSA-（SS 14）（3）的表达水平远低于（SS 14）（2）-HSA。基质辅助激光解吸电离飞行时间质谱分析表明，在发酵过程中发生了严重的降解。与生长抑素-14的标准相似，所有三种融合蛋白都能够抑制血液中生长激素的分泌，其中（SS 14）（2）-HSA是最有效的一种。总体而言，（SS 14）（2）-HSA在产量和生物活性方面都是最有效的蛋白，增加与HSA融合的小拷贝数可能不是提高蛋白生物活性的合适方法。
Somatostatin is a natural inhibitor of growth hormone, and its analogues are clinically used for the therapy of acromegaly, gigantism, thyrotropinoma, and other carcinoid syndrome. However, natural somatostatin is limited for clinical usage because of its short half-life in vivo. Albumin fusion technology was used to construct long-acting fusion proteins, and Pichia pastoris was used as an expression system. Three fusion proteins, (somatostatin (SS)14)(2)-human serum albumin (HSA), (SS14)(3)-HSA, and HSA-(SS14)(3), were constructed with different fusion copies of somatostatin-14 and fusion orientations. The expression level of (SS14)(3)-HSA and HSA-(SS14)(3) was much lower than (SS14)(2)-HSA due to the additional fusion of the somatostatin-14 molecule. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry revealed that severe degradation occurred in the fermentation process. Similar to the standard of somatostatin-14, all three fusion proteins were able to inhibit growth hormone secretion in the blood, with (SS14)(2)-HSA being the most effective one. On the whole, (SS14)(2)-HSA was the most effective protein in both production level and bioactivity, and increasing the number of small protein copies fused to HSA may not be a suitable method to improve the protein bioactivity.