RECEPTOR
2.1 Introduction
The Epidermal Growth Factor Receptor is a 170 KDa protein containing an extracellular domain, a transmembrane region and an intracellular tyrosine kinase domain. The ligands of EGFR eg. EGF binds to the extracellular region within the amino-terminal of 622 amino acids. The Extra cellular region/ domain (ECD) is further sub devided into four domains I–IV, also known as the L1, S1, L2, and S2 domains, respectively (Bajaj et al., 1987). Domains I and III share 37% amino acid identity, while domains II and IV are homologous Cys-rich domains, CR1 and CR2, respectively (Ward et al., 1995). The ligand-binding domain of the epidermal growth factor receptor is separated from the cytoplasmic protein tyrosine kinase domain by a single transmembrane domain of 24 amino acids (Carpenter et al., 1991). The intracellular portion of the epidermal growth factor receptor consists of a tyrosine kinase domain of 541 amino acids (Hanks et al., 1988) which is further subdivided into a core kinase domain of approximately 290 amino acids and a COOH-terminal domain of approximately 230 amino acids. The COOH- terminal domain contains all five sites of autophosphorylation (Cadenas et al., 1994;
Downward et al.; Margolis et al., 1989; Walton et al., 1990) which plays an important role in the assembly of substrates involved in signal transduction through SH2 domain (Cantley et al., 1991; Koch et al., 1991). The COOH terminus also contains sequences required for ligand-induced endocytosis and down-regulation of cell surface receptors (Chang et al., 1993, 1991; Chen et al., 1989; Wiley et al., 1991).
Fabricant et al established the expression of EGFR protein in the placenta and A431 carcinoma cell lines (Fabricant et al., 1977). Initially A431 cell lines were chosen for the isolation of EGFR because they expresses abnormally high levels of EGFR viz 10-50 times more than any other cell lines (Fabricant et al., 1977; Wrann and Fox, 1979). In the pioneering studies by Cohen, intact detergent solubilized membrane were isolated from A 431 cell line which retain the ability to bind to ligand EGF (Cohen et al., 1981).
Their group established the biochemical mechanisms of EGFR signaling by working with detergent solubilized preparation of EGFR. Furthermore, partial purification of endogenously expressed EGFR from A 431 cells was achieved by immunoaffinity
chromatography and other means (Cohen et al., 1982, 1981; Hock et al., 1980; Weber et al., 1984; Yarden et al., 1985). While these approaches provided sufficient quantities of wild-type protein preparations for biochemical characterization, but they do not provide sufficient quantities or a means to isolate mutant constructs for biophysical characterization of intact receptor molecules. In addition, the stability of highly purified detergent-soluble EGFR preparations has not been characterized in detail. Later on for the production of EGFR protein in large quantity, EGFR expressing cell lines such as A 431, CHO cell line or placenta were used for the construction of EGFR cDNA. For example the cDNA from placenta and A431 cell line were used for the cloning of EGFR using λg10 vector system (Ullrich et al., 1984). Sato et al isolated full-length cDNA encoding EGFR by PCR from a human placenta and cloned into mammalian expression plasmid pcDNA3.1/Zeo (+) (Sato et al., 2000). Lin et al constructed prokaryotic expression plasmid pET30a (+) carrying intracellular domain of EGFR from the mRNA of A 431 cell line. The recombinant protein was purified using Ni2+-NTA agarose for the generation of monoclonal antibodies (Lin et al., 2004). In another study the recombinant intracellular domain were produced as a glutathione S-transferase (GST) fusion in Escherichia coli using pGEX6-P-1 vector, and the solubilisation was performed by sarkosyl addition during extraction (Elloumi-Mseddi et al., 2013). The ectodomain of EGFR was cloned, expressed and purified using polycistronic approach. This expression system yielded 20 fold higher than that of A 431 cell line (Cadena and Gill, 1993).
Generally for the purification of recombinant eukaryotic protein mammalian expression system or baculovirus system are used. Many studies were reported where mammalian expression system (Cadena and Gill, 1993; Chen et al., 2008; Mi et al., 2008; Ogiso et al., 2002) or insect cell lines (Ferguson et al., 2003; Greulich et al., 2005) were used for the expression and purification of extra cellular domain of EGFR, which is more expensive compared to E. coli. The advantages of E. coli as an expression host include well studied physiology, genetics and availability of advanced genetic tools, rapid growth, high-level protein production rates achieving up to 10–30% of total cellular protein, ease of handling in a standard molecular biology laboratory, low cost and the ability to multiplex both expression screening and protein production. There are however several disadvantages, particularly for eukaryotic proteins, of expression in a prokaryotic
system. The lack of eukaryotic chaperones, specialised post-translational modifications, ability to be targeted to sub-cellular locations or to form complexes with stabilising binding partners can result in protein mis folding and aggregation.
In the present study EGFR was chosen as a target for the development of oligonucleotide probes that can be used for the detection of EGFR. Structurally EGFR is devided into three domains, viz Extra cellular Domain, Transmembrane domain and cytoplasmic intracellular domain. The extra cellular domains plays most significant role as it is involved in binding of its ligands and further stimulation of various signalling pathways.
Thus extra cellular domain i.e. ECD of EGFR was cloned, expressed and purified for aptamer selection. The full length EGFR gene cloned in pBABE vector was procured from Addgene plasmid repository. The ECD region of EGFR was subcloned into pET 28a (+) vector and expressed in Escherichia coli BL21 strain. The expressed proteins were further purified by immobilized metal ion affinity chromatography (IMAC) and confirmed by western blot analysis. The ECD region of EGFR proteins were purified for the selection of EGFR specific aptamers.
Figure. 2.1: Molecular architecture of EGFR showing boundaries and designation of different domains.