4.2.1 Choice of plasmid and cell lines
The triple resonance experiments conducted in this study required that an efficient protocol be devised for expressing high yields of PDGF R-TM. This is primarily because high costs of isotopic labels can render the study infeasible if good yields are not obtained. A single NMR sample of 180µL requires a protein concentration of at least 0.3 mM for 3D analyses and it is of utmost importance to maximise yields to make it economically feasible.
Previous work by Dr Joanne Oates to characterise the role of TM and JM domains in oligomerisation of PDGF R involved the construction of plasmids where PDGF R- TM with either 20, 30 or 40 residues on either side were cloned into a pET30a vector (Novagen) for expression in E. coli (Oates et al., 2010). All of these vectors contained a His-tag which allowed for purification using IMAC. These constructs will henceforth be referred to as pET30a-PDGF R-TM-20, pET30a-PDGF R-TM- 30 and pET30a-PDGF R-TM-40 respectively. It was thought that these constructs would form an ideal starting point for the expression of PDGF R-TM for NMR analyses.
Cells were grown directly from glycerol stocks which had the constructs of inter- est in 3 di↵erent types of E. coli cell lines: BL21(DE3)pLysS; C41; and C43. BL21(DE3)pLysS cells allow protein expression of any gene that is under the control of a T7 promoter and has a ribosome binding site. These cells also contain a pLysS plasmid which carries the gene encoding T7 lysozyme which lowers ‘leaky’ expression but does not interfere with the level of expression achieved following induction by iso- propyl -D-1-thiogalactopyranoside (IPTG). C41 cells are derived from BL21(DE3),
and contain a mutation which prevents cell death associated with recombinant ex- pression of some proteins which may be toxic to the cells. C43 cells are a derivative of C41 which can express a di↵erent set of toxic proteins than C41.
The C41 and C43 cells did not grow in LB medium in the presence of antibiotics even after several attempts. The transformed BL21(DE3)pLysS cells grew in the presence of antibiotics. The plasmid was extracted and sent for sequencing to verify that no mutations occurred in the sequence.
4.2.2 Expression of pET30a-PDGF R-TM-40
Prior to optimising conditions for expression in minimal media, the pET30a-PDGF R- TM-40 plasmid was transformed into BL21(DE3)pLysS cells (see section 2.5.3) and expressed in LB (see section 2.6.1) as per existing protocols (Oates et al., 2010) to check for expression levels. Once the cells were harvested, a portion of the whole cells (WC) were lysed and centrifuged. The resulting supernatant (S) and pellet (P), along with the whole cells were analysed on an SDS-PAGE gel as well as a western blot as detailed in sections 2.8.1 and 2.8.4. The expected molecular mass of the expressed protein is 18.1 KDa and the gel (Figure 4.2a) revealed a thick band at the position of the expected monomer in the whole cell as well as pellet fractions (highlighted in red box in panel (a)). This suggested that the protein was being expressed in inclusion bodies. Inclusion bodies are formed for 70% of recombinant proteins over-expressed inE. coli (Yang et al., 2011). This is usually due to the lack of eukaryotic chaperones and the saturation ofE. coli post-translational machinery (Singh et al., 2015).
The western blot (Figure 4.2b) corroborates that the bands observed are of PDGF R- TM-40 as only proteins with a His-tag will show up on the blot. A dimer is also observed at about 40 kDa. This is slightly more than the expected 36.2 kDa but such anomalous migration has frequently been reported in literature for membrane proteins, especially for higher order oligomers (Rath et al., 2009). In particu- lar, slow migration of higher order oligomers of PDGF R-TM has been observed
(a) SDS-PAGE (b) Western blot
Figure 4.2: Expression pET30a-PDGF R-TM-40 in LB. Panel (a) shows the SDS- PAGE gel whereas B shows the anti-His western blot. Cells were grown at 37 for 4 hours after induction with 1 mM IPTG. Red box highlights the position of the expected monomer PDGF R-TM-40 band. WC: whole cells, S: supernatant after lysis and centrifugation of whole cells, P: pellet after lysis and centrifugation of whole cells.
previously (Oates et al., 2010).
Once good expression levels were observed in rich medium, the PDGF R-TM-40 protein was expressed in M9 minimal media. The SDS-PAGE gel shown in Fig- ure 4.3a reveals that even though the expression levels drop considerably in minimal medium, clear bands corresponding to the protein were seen (highlighted in red box in panel (a)). A anti-His western blot confirmed this and a dimer of the protein was once again observed (Figure 4.3b). A lane of non-induced whole cells (N) was run on both SDS-PAGE and western blots to ensure there was no ‘leaky’ expression and that the cells weren’t natively producing a protein which was being bound by anti-His antibody during western blotting.
(a) SDS-PAGE (b)Western blot
Figure 4.3: Expression pET30a-PDGF R-TM-40 in M9 minimal media. Panel (a) shows the SDS-PAGE gel whereas B shows the anti-His western blot. Cells were grown at 37 for 4 hours after induction with 1 mM IPTG. Red box highlights the position of the expected monomer PDGF R-TM-40 band. WC: whole cells, S: supernatant after lysis and centrifugation of whole cells, P: pellet after lysis and centrifugation of whole cells, N: non-induced whole cells.
4.2.3 Comparison of expressions with varying JM lengths
As mentioned previously, the juxtamembrane regions are known to play an impor- tant role in the activation and oligomerisation of the PDGF R. It was observed by Oates and coworkers (Oates et al., 2010) that the PDGF R-TM on its own forms higher order oligomers in vitro which are not biologically relevant. It was further observed that upon the addition of 40 juxtamembrane residues on either side, the peptide existed exclusively as monomers and dimers.
Since the goal for this study was to prepare samples for NMR analyses, it was preferred that the number of JM amino acids to be included in the sample be min- imised so as to avoid spectral crowding. This is especially important in the case of a membrane protein embedded in detergent micelles which can lead to broad peaks in an NMR spectrum. Figure 4.4 shows a western blot of the expression levels of pET30a-PDGF R-TM-40 (JM40), pET30a-PDGF R-TM-30 (JM30) and pET30a- PDGF R-TM-20 (JM20) in M9 minimal medium. Whole cells (WC) as well as
sion levels were observed for the smallest protein, PDGF R-TM with 20 resides on either side of the TM. For these reasons, all further work was carried out using this construct.
Figure 4.4: Expression levels of pET30a-PDGF R-TM with varying JM lengths observed using an anti-His western blot. Cells were grown at 37 in M9 medium for 4 hours after induction with 1 mM IPTG. WC: whole cells, P: pellet after lysis and centrifugation of whole cells, M: protein ladder, JMX: PDGF R-TM with X residues on either side.