Recombinant production of human ICAM-1 chimeras by single step on column refolding and purification

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• 作者：David Nú ; ñ ; ez^a ; ^b ; Marí ; a Pilar Domingo^a ; ^b ; Diego Sá ; nchez-Martí ; nez^b ; ^c ; Vicente Cebolla^a ; Arthur Chiou^d ; Adriá ; n Velá ; zquez-Campoy^c ; ^e ; ^f ; Juliá ; n Pardo^b ; ^c ; ^f ; ^g ; ¹ ; Eva Ma Gá ; lvez^a ; ^b ; ¹ ; ^{eva@icb.csic.es}
• 关键词：Adhesion molecules ; On-column refolding ; LFA-1
• 刊名：Process Biochemistry
• 出版年：2013
• 出版时间：April, 2013
• 年：2013
• 卷：48
• 期：4
• 页码：708-715
• 全文大小：1570 K

文摘

The interaction of the adhesion molecule of the immunoglobulin family intercellular adhesion molecule 1 (ICAM-1) with its ligands such that the integrins LFA-1 and Mac-1 is crucial for the regulation of several physiological and pathophysiological processes like cell mediated-elimination of tumor or virus infected cells, cancer metastasis or inflammatory autoimmune processes. Thus, production of milligrams of protein is required to perform structural and functional studies as well as design novel approaches to find out new inhibitors of ICAM-1/LFA-1 interaction. Here we report on the production of a recombinant human ICAM-1 chimera comprising the first two extracellular domains of ICAM-1 linked to the Fc fraction of a human IgG1. To this aim we have used a cost-effective method based on the expression of a His-tagged protein in Escherichia coli followed by a single step of refolding and purification on immobilized metal affinity columns. This method is able to produce 3 mg/l of bacterial culture in just 72 h with purity greater than 95 % . The identity and the native structure of refolded human ICAM-1 chimera were confirmed by biochemical and biophysical studies including SDS-electrophoresis, immunoblot, circular dichroism, isothermal titration calorimetry and fluorescence spectroscopy. Native folding and functional activity of the chimera were further confirmed by different cell biology studies, including B cell adhesion, T cell binding and inhibition of NK cell function. These studies indicate a high biological activity of the protein since it induces a 200-fold increase/mg of protein in B cell adhesion and the inhibitory dose 50 to block cell-mediated cytotoxicity is 10 pg/effector cell. These analyses show that our protocol is able to produce a recombinant human ICAM-1 chimera fully active and useful to analyze the biological processes in which ICAM-1/LFA-1 interaction is critically involved.