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

编码干扰素-α的整个胞外区的基因 受体(缺少信号肽)已克隆到pGEX-2T中 并在大肠杆菌DH5中以细菌融合蛋白的形式表达 谷胱甘肽-S-转移酶。编码28-437个氨基酸的DNA片段 将受体蛋白插入到载体pGEX-2T的EcoRI处， BamHJ网站。紧邻受体下游的终止密码子 添加了序列。IPTG诱导表达，融合蛋白 Sepharyl P S-300凝胶柱层析部分纯化蛋白质 然后用谷胱甘肽琼脂糖亲和层析。工作开始了 修复非融合受体后蛋白水解酶的研究进展 凝血酶，并启动对凝血酶的理化和生物学研究 重组蛋白的鉴定；抗血清 将产生受体并用于纯化天然蛋白 并将作为研究其机制的探针 涉及干扰素-α及其受体的信号转导(S)。

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).