RECEPTOR
2.4 Discussions
The extra cellular domain of EGFR plays most significant role as it binds to ligand and induces the activation of downstream signaling pathway. Also these domains are responsible for the formation of either homodimers or heterodimers that amplifies the signalling cascades. Mostly alongwith the full length EGFR, mutant variants of EGFR are also detected in normal and malignant cells, in tissues and in biological fluids of cancer patients (Perez-Torres et al., 2008; Rose-John and Heinrich, 1994). These isoforms/variants are comprised solely of extra cellular domain portions of the receptor.
It is very important that any probe developed for the detection of EGFR should be directed to extra cellular domain of EGFR so that it can detect all the EGFR variants alongwith the full length EGFR. Thus in the present study the extra cellular domain of EGFR protein was cloned, expressed and purified for aptamer selection.
The main objective of this chapter was to subclone EGFR Extra cellular Domain into pET28a vector and purify the EGFR ECD protein in non-denaturing condition for aptamer selection. The full length EGFR cDNA cloned in pBABE vector in DH5α strain was procured from Addgene plasmid repository (Plasmid # 11011). Then the ECD region of EGFR was PCR amplified from EGFR WT plasmid using ECD_F1 and ECD_R1 primer. The primers were prefabricated with restriction enzyme site. This strategy is simple and still the most common, method for cloning PCR products. It allows direct, directional cloning of the insert into the vector after restriction digestion (Kaufman and Evans, 1990). Further to improve the efficiency of cloning of ECD into pET28a vector, the PCR amplicons were first cloned into pTZ57R/T vector for TA cloning. The TA cloning method takes advantage of the terminal transferase activity of Taq polymerase.
This enzyme adds a single, 3'-A overhang to each end of the PCR product. This makes it possible to clone the PCR product directly into a linearized cloning vector with single 3'- T overhangs. Also the colony screening for positive clones can be easily performed by blue-white screening. The colonies carrying recombinant vector produces white colonies, whereas blue colonies usually do not contain any clone.
For the cloning and expression of recombinant ECD of EGFR protein, pET-28a (+) vector system was selected. pET28a (+) vector is a most commonly used vector for cloning and expression of recombinant proteins in E. coli. It is a modified form of pBR322 with a strong T7 promoter system originally developed by Studier and colleagues (Studier et al., 1990; Studier and Moffatt, 1986). The desired genes to be cloned in pET plasmids are under the influence of strong bacteriophage T7 transcription and the expression is induced by T7 RNA polymerase in the host cell. Although this system is extremely powerful, it allows the user to control the expression levels simply by manipulating the concentration of inducer. Another advantage with pET system is its ability to maintain target genes transcriptionally silent in the uninduced state. Also the His6-Tag are incorporated at the ends of recombinant protein. The rationale for the choice of an N-terminal hexahistidine is manifold. An N-terminal tag ensures that the bacterial transcription and translation machineries always encounter 5′ and N-terminal sequences that are compatible with robust RNA synthesis and protein expression, respectively. The incorporation of oligo histidine-tagged dramatically aid in proteins purification using a relatively simple protocol of immobilized metal affinity chromatography (IMAC).
Further, histidine tags rarely affect the characteristics, which distinguishes it, for example, from glutathione S -transferase (GST), which itself is a dimer that then imposes dimerization on the recombinant protein. Also hexahistidine tags do not have a consistent impact on the N-terminal structure of the target protein.
The recombinant pET-28a (+) vector carrying EGFR ECD were transformed into E. coli (BL21) cells for the production of recombinant protein. The E. coli as a host is the most widely used system for protein overproduction, both on a laboratory and industrial scale.
It has the advantage of producing large quantities of recombinant proteins in a short time.
A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. Over the years, much effort has been put into optimizing E. coli as an expression host for production of stably folded proteins from higher organisms (Peti and Page, 2007). BL21 (DE3) strain of E. coli is an appropriate E. coli strain for high-level protein production purposes. It has the advantage of being deficient in both lon and ompT proteases and it is compatible with the T7 lacO promoter system (William Studier et al., 1990). For eukaryotic
proteins, it is often important to use BL21 (DE3) derivatives carrying additional tRNAs to overcome the effects of codon bias. Another crucial factor of protein production is the temperature during induction. Generally in T7 system many recombinant proteins often precipitate when expressed at 37 °C, but are soluble when the temperature during induction is 15–25 °C, presumably because slower rates of protein production allow newly transcribed recombinant proteins time to fold properly (Vera et al., 2007). Thus, lower temperature was used in our study for expression of ECD region of EGFR protein so that the recombinant protein folds properly. Still the major drawback of this system is inefficient post-translational modification mechanism in E. coli. Here in this study ECD region of protein was purified for the aptamer selection. The selected aptamers were used explored as a biorecognition probe in various assays of biomedical research. Thus to ascertain that the selected aptamers were able to recognize the native conformation of EGFR, different cancerous cell lines were used for binding studies.