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首页> 外文期刊>Protein Expression and Purification >Expression, purification, and characterization of recombinant human serum albumin fusion protein with two human glucagon-like peptide-1 mutants in Pichia pastoris
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Expression, purification, and characterization of recombinant human serum albumin fusion protein with two human glucagon-like peptide-1 mutants in Pichia pastoris

机译：带有两个人胰高血糖素样肽-1突变体的重组人血清白蛋白融合蛋白的表达，纯化和鉴定

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摘要

Glucagon-like peptide-1 (GLP-1)is a 30-residue peptide hormone secreted by intestinal L-cells in response to nutrient ingestion. In the present study, overlapping PCR technology was employed to construct two GLP-1 mutants (GLP-1(A2G))(2) and human albumin (HSA) genes in vitro without linker. The spliced gene, (GLP-1(A2G))(2)-HSA, was over expressed under the control of promoter AOX1 and Mat alpha signal peptide in Pichia pastoris. SDS-PAGE and Western blotting were applied to assay the recombinant fusion protein in the culture broth. The results demonstrated that the recombinant (GLP-1(A2G))(2)-HSA concentration in the broth could reach a level of 245.0 mg/L and the expressed fusion protein was capable of cross-reacting with anti-human GLP-1 and anti-human albumin antibody. The recombinant (GLP-1(A2G))(2)-HSA protein was purified by ultrafiltration, columns of Q-sepharose fast flow and Superdex 75 size-exclusion. The recombinant (GLP-1(A2G))(2)-HSA protein obtained could lower in vivo glucose concentration in blood and stimulate in vitro islet cell proliferation. In mouse model, the fusion protein was detectable in plasma even 308 h after a single subcutaneous dose of 1.25 mg/kg. The result showed that the terminal biological half-time of the protein was about 54.2 h which is 650-fold longer than that of GLP-1. The pharmacokinetic analysis of the protein suggests its promising application in clinical medicine. (C) 2008 Elsevier Inc. All rights reserved.
机译：胰高血糖素样肽1（GLP-1）是一种由肠L细胞分泌的30个残基的肽激素，可响应营养摄入。在本研究中，重叠PCR技术用于构建两个没有连接子的体外GLP-1突变体（GLP-1（A2G））（2）和人白蛋白（HSA）基因。剪接的基因，（GLP-1（A2G））（2）-HSA，在巴斯德毕赤酵母中在启动子AOX1和Mat alpha信号肽的控制下过表达。应用SDS-PAGE和蛋白质印迹法测定培养液中的重组融合蛋白。结果表明，肉汤中的重组（GLP-1（A2G））（2）-HSA浓度可达到245.0 mg / L，表达的融合蛋白能够与抗人GLP-1交叉反应。和抗人白蛋白抗体。重组（GLP-1（A2G））（2）-HSA蛋白通过超滤，Q-琼脂糖快速流动色谱柱和Superdex 75尺寸排阻色谱纯化。获得的重组（GLP-1（A2G））（2）-HSA蛋白可降低血液中的体内葡萄糖浓度并刺激体外胰岛细胞增殖。在小鼠模型中，即使在单次皮下注射1.25 mg / kg后308小时，血浆中仍可检测到融合蛋白。结果表明，该蛋白质的最终生物学半衰期约为54.2小时，是GLP-1的650倍。对该蛋白质的药代动力学分析表明其在临床医学中的应用前景广阔。 （C）2008 Elsevier Inc.保留所有权利。

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