Expression, purification, and characterization of rat interferon-β, and preparation of an N-terminally PEGylated form with improved pharmacokinetic parameters
Expression, purification, and characterization of rat interferon-β, and preparation of an N-terminally PEGylated form with improved pharmacokinetic parameters
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DOI：
10.1016/j.pep.2003.11.004
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发表时间：
2004-04-01
影响因子：
1.6
通讯作者：
Baker, DP
中科院分区：
文献类型：
--
作者：
Arduini, RM;Li, ZF;Baker, DP
为了确定人干扰素-β-1a的潜在临床用途和给药途径，我们开发了一种高纯度的大鼠干扰素-β的表达和纯化程序，适合于在人类疾病的大鼠模型上进行测试。构建了含有大鼠干扰素-β信号序列和结构基因的表达载体，并将其导入中国仓鼠卵巢细胞。用标准层析技术从CHO细胞条件培养液中纯化得到纯度为99.5%的蛋白。分析表明，该蛋白是一种高度糖基化的单体蛋白，预测的四个N-糖基化位点中有两个占据了该蛋白。对所附低聚糖的分析表明，它们是双天线、三天线和四天线结构的复杂混合物，其中以唾液酸化的三天线和四天线结构为主。肽图谱、N-末端测序和质谱分析证实了纯化蛋白的真实性和完整性。纯化的蛋白在大鼠RATEC细胞上的比活性为2.1×10(8)U/mg，与小鼠和人干扰素-β1a在小鼠和人细胞上的效价相近。我们还制备了一种N-末端聚乙二醇化的大鼠干扰素-β，其中20 kDa的甲氧基聚乙二醇丙醛连接到Ile-1的N-末端α-氨基上。与未修饰的蛋白相比，聚乙二醇化的蛋白在体外基本上保持了完全的抗病毒活性，并改善了大鼠的药代动力学参数。未经修饰和聚乙二醇化的两种形式的大鼠干扰素-β都将用于人类疾病的大鼠模型的测试。(C)2004 Elsevier Inc.保留所有权利。
To identify potential new clinical uses and routes of administration for human interferon-beta-1a (IFN-beta-1a), we have developed an expression and purification procedure for the preparation of highly purified rat interferon-beta (IFN-beta) suitable for testing in rat models of human disease. An expression vector containing the rat IFN-beta signal sequence and structural gene was constructed and transfected into Chinese hamster ovary (CHO) cells. The protein was purified from CHO cell conditioned medium and purified to >99.5% purity using standard chromatographic techniques. Analytical characterization indicated that the protein was a heavily glycosylated monomeric protein, with two of the four predicted N-glycosylation sites occupied. Analysis of the attached oligosaccharides showed them to be a complex mixture of bi-antennary, tri-antennary, and tetra-antennary structures with a predominance of sialylated tri-antennary and tetra-antennary structures. Peptide mapping, N-terminal sequencing, and mass spectrometry confirmed the identity and integrity of the purified protein. The purified protein had a specific activity of 2.1 x 10(8) U/mg when assayed on rat RATEC cells, which is similar in magnitude to the potencies observed for murine IFN-beta and human IFN-beta-1a assayed on murine and human cells, respectively. We also prepared an N-terminally PEGylated form of rat IFN-beta in which a 20 kDa methoxy polyethylene glycol (PEG)propionaldehyde was attached to the N-terminal alpha-amino group of Ile-1. The PEGylated protein, which retained essentially full in vitro antiviral activity, had improved pharmacokinetic parameters in rats as compared to the unmodified protein. Both the unmodified and PEGylated forms of rat IFN-beta will be useful for testing in rat models of human disease. (C) 2004 Elsevier Inc. All rights reserved.