融合蛋白(GLP-1_(A2G))_2-HSA的分离纯化及其活性评价

Purification and Identification of the Activity of Fusion Protein(GLP-1_(A2G))_2-HSA

建立简便、快速、高效的重组人血清白蛋白与胰高血糖素样肽-1(GLP-1)突变串联体融合蛋白(GLP-1A2G)2-HSA(GGH)的分离纯化方法。重组毕赤酵母菌株发酵产生的融合蛋白GGH的上清液通过超滤、染料配体亲和层析、凝胶过滤层析、离子交换层析获得高纯度的样品,终回收率为33.1%,12%SDS-PAGE和HPLC检测纯度达到98%。体内活性测定结果表明,融合蛋白GGH在小鼠皮下给药8 h后仍能发挥显著的降血糖作用。结果表明建立的分离纯化方法较好的保留了融合蛋白(GLP-1A2G)2-HSA的生物活性。

Glucagon-like peptide-1（GLP-1） is a 30-residue peptide hormone secreted by intestinal L-cells in response to nutrient ingestion.A fusion protein（GLP-1A2G）2-HSA） of two human glucagon-like peptide-1 mutant（GLP-1A2G） and human serum albumin（HSA） was expressed and secreted into the fermentation broth with recombinant Pichia pastoris.Inorder to establish a simple and efficient method of purification for the fusion,the（GLP-1A2G）2-HSA（GGH） was purified from fermentation broth by centrifugation,ultrafiltration concentration,affinity absorption chromatography,gel filtration and ion exchange chromatography.The final products were conformed as one single band by SDS-PAGE and the purity was identified as 98%.The total recovery yield could reach up to 33.1%.Furthermore,the analysis of activity in vivo indicated that GGH could significantly reduce blood glucose level after subcutaneous injection 8 h.It suggested that the purification method kept the high activity of GGH and promised the half-time of GLP-1.