EXPRESSION AND ISOLATION OF A FUNCTIONAL IFN ALPHA RECEPTOR FROM INCLUSION BODIES

功能性 IFN α 受体的表达和从包涵体中的分离

• 批准号: 3792480
• 负责人: N Y NGUYEN
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3792480
• 关键词: antireceptor antibody; biological signal transduction; chimeric proteins; cytokine receptors; inclusion body; interferons; protein purification; protein structure function; receptor expression

The gene coding for the entire extracellular domain of the IFN-alpha receptor (lacking the signal peptide) has been cloned in plasmid pGEX-2T and expressed in E. coli DH5 as a fusion protein with bacterial glutathione-S-transferase. A DNA fragment encoding amino acids 28-437 of the receptor protein was inserted into vector pGEX-2T at the EcoRI and BamHJ sites. A stop codon immediately downstream of the receptor sequence was added. Expression was induced by IPTG and the fusion protein was partially purified by gel chromatography on Sepharyl P S-300 followed by glutathione agarose affinity chromatography. Work is in progress to recover the non-fused receptor after-protease cleavage by thrombin, and to initiate studies on the physico chemical and biological characterization of the recombinant protein; antisera against the receptor will be produced and will be used to purify the natural protein from mammalian cells, and will serve as a probe to study the mechanisms of signal transduction involving interferon-alpha and its receptor(s).

编码IFN-α的整个胞外区的基因 已将受体（缺少信号肽）克隆到质粒pGEX-2 T中 转化大肠大肠杆菌DH 5作为融合蛋白与细菌 谷胱甘肽-S-转移酶 一种编码SEQ ID N 0：28-437的氨基酸的DNA片段， 将受体蛋白在EcoRI处插入载体pGEX-2 T中， BamHJ网站。 受体下游的终止密码子 序列被添加。 IPTG诱导表达，融合蛋白 通过Sepharyl PS-300凝胶层析部分纯化蛋白质 随后进行谷胱甘肽琼脂糖亲和层析。 工作正在 蛋白酶切割后回收非融合受体的进展 凝血酶，并启动物理化学和生物学研究 重组蛋白的表征;抗 受体将被产生，并将用于纯化天然蛋白质 从哺乳动物细胞，并将作为一个探针，研究机制 干扰素-α及其受体参与的信号转导。