Purification and functional analysis of the recombinant protein isolated

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Abstract: In this paper, the purification of the human recombinant protein expressed in E. coli using the GST Gene Fusion System, by applying various methods of bacterial lysis: sonication, freeze/thaw and beadbeating, is presented. The study was an attempt to compare the properties of the proteins obtained by the sonication method, recommended by manufacturers but inaccessible for many researchers, with those obtained using two other readily available lysis methods. The data show that all purified proteins were soluble and intact with the highest protein yield being obtained via the freeze/thaw method. The results of functional analysis indicate that the proteins purified using the sonication and freeze/thaw methods of lysis exhibited similar DNA binding affinity, while the protein purified by beadbeating was also functional but with a lower binding affinity. The conclusion of this study is that all three lysis methods could be successfully employed for protein purification. Keywords: protein purification, bacterial lysis, protein yield, protein-DNA interaction. The synthesis of recombinant proteins is essential for any biochemical study on gene control. Although in vivo experiments have powerful applications, most of the key molecular details of the action of a protein have to be resolved biochemically. Therefore, recombinant proteins or their important regulatory domains are synthesized and studied in vitro. Escherichia coli expression systems are the most efficient, most commonly used and inexpensive model systems for the synthesis of intact, folded recombinant proteins under 75 kDa.1 There are many methods that can be employed to overproduce a recombinant protein in E. coli. One of them is the addition of sequence tags to a protein. In this strategy,the DNA fragment encoding the gene of interest is cloned into a vector containing an in-frame purification tag.1 The common purification tags fused to eukaryotic proteins expressed in E. coli systems are His6, glutathione-S-transferase (GST), and maltose-binding protein (MBP), although many others have been successfully used.1 In this work, the employment of the GST Gene Fusion System (Amersham Pharmacia Biotech) for overexpression and subsequent purification of the human recombinant protein from E. coli is described. This fusion system is the most commonly used method since it has many advantageous characteristics: stabilizing effect on some proteins, enhancement of fusion protein expression in E. coli and aiding protein solubilization.1 Retinoic acid (RA), a derivative of vitamin A, is important for the maintenance of normal cell growth, differentiation and development.2 One route of its action is mediated through activation of retinoid X receptors (RXRs),3 which then bind to response elements within the promoter region of RAtarget genes, thus regulating their expression.4–6 The purification of human RXR protein using the GST System by applying three different ways of bacterial lysis, this being one of the crucial steps during purification, is described herein. The aim was to compare the proteins obtained by the sonication method, which is recommended by manufacturer but is not available for many researchers, with those obtained by the two other lysis methods, usually on hand in many laboratories, i.e., freeze/thaw and beadbeating. Lysis by sonication requires not only expensive apparatus but also lengthy optimization of the working conditions. The results of this study show that the sonication method could be successfully replaced by the other two lysis methods.