42
43 Expression of soluble, recombinant VP7 was successful. However, the expected 34 kDa VP7 (37 kDa – including the leader peptide) was not visible. Instead, a 27 kDa protein was expressed and observed in all samples. Sequence analysis of the pCOLDI_VP7-ER vector indicated that during the transformation, a 159 bp fragment of the ORF (bp 365-524) encoding for 53 amino acids (AA 121-174) was deleted (Figure 15 B). This deletion spans the variable regions VR6 and VR7 of VP7. This truncation of the proteins disrupted two disulphide bonds in the protein's tertiary structure (Aoki et al., 2009) (Figure 15 B). Although the protein produced from this experiment was incomplete, its immunogenic epitopes were still intact and could be used in future work to establish an immunogenic response specific to RV_GR10924 VP7. See appendix for pCOLDI_VP7-ER sequencing data.
Figure 15: A. CryoEM structure of the complete virion 25 Å resolution. B. Schematic diagram of VP7 primary structure, including the signal sequence (residues 1-50, light gray). The two domains are in the same colours as in A; The pattern of the intrasubunit disulphide bonds with numbers corresponding to the positions of the cysteine residues.
C. Ribbon diagram of the VP7 trimer (left), with one subunit and one pair of Ca2+ ions in colour. The Rossmann-fold domain (domain I) is in yellow; the β-barrel domain (domain II), in orange; the Ca2+ ions, in blue. A single subunit, inside view, is shown on the right, with secondary structure elements labelled (Aoki et al., 2009). With permission from publishers.
44 2.3.2 Rotavirus VP2 and VP6 expression
For a previous experiment, the plasmid pET28_GR10924_VP2 was constructed and purchased from GeneArt™ (Mr JJ van Greunen). The GR10924_VP2 plasmid was used for co-expression with pET_GR10924_VP6, purchased from GeneArt™, and thus was put under a different antibiotic resistance control (kanamycin selection). (See appendix Figure 37, Figure 39).
Various expression conditions were utilised for the expression of RV VP2/VP6 DLPs in bacteria. These conditions included using different expression cell lines, different IPTG concentrations for induction of expression, various expression temperatures (using thermo-sensitive induction promoters), induction times, chaperones, and different expression media.
Expression cell lines listed in Table 7 illustrate the various features that have been exploited during this experiment. As described before, in Section 2.2.1, Tuner cells were used to slow the expression of recombinant proteins through the regulation of the IPTG concentration. The expression can be regulated from deficient levels to robust, fully induced levels commonly associated with pET hosts. The lower-level expression may enhance the solubility of toxic proteins or proteins with complicated secondary structures. IPTG concentration can be adjusted between 0.1 mM to 4 mM. Temperatures of 20°C, 30°C, and 37°C have been used to change the bacteria's metabolic rate, which aids in folding the desired proteins, further increasing their solubility and reducing their toxicity (Vera et al., 2007).
The optimal induction time of the protein in bacterial cells is 4 hours, according to the manufacturer’s manual for the pET vector. Overnight induction can lead to the depletion of the antibiotics used for the selection of recombinant cells (Novagen, 2003). This, in turn, might lead to increased representation of non- recombinant cells, thus lowering expression efficacy. With this drawback in mind, overnight expression was still attempted to increase overall protein yield. Lower temperature overnight expression was also attempted to overcome the depletion of resources previously described (Novagen, 2003).
45 Additionally, the folding of proteins was modulated through the incorporation of various chaperones. Although the protein's amino acid sequence contains information needed to adapt the protein's tertiary structure, some proteins are misfolded due to environmental stresses and the lack of eukaryotic co-factors.
The chaperone set pGro7 (GroEL/GroES), induced with L-arabinose, was used to prevent aggregation of insoluble proteins and aid in forming soluble proteins (Figure 16).
Figure 16: SDS-PAGE analysis of VP2 and VP6 in Origami cells with GroEL/GroES chaperones. TF: Total fraction, SF: Soluble fraction. GroEL molecular weight in its monomeric form is 60 kDa and is visible in lanes 4, 6, and 8. GroES molecular weight is 10 kDa but can be seen here at around 14 kDa in both the TF and SF. Neither VP2 nor VP6 expression can be observed. Lane 2 and 3 indicated no expression of the chaperones as expected in the uninduced control. Lane 4 and 5 show chaperone expression in the induced control. Lane 6 and 7 reveal the GR10924_VP2 with chaperones. No expression for VP2 at 102 kDa can be observed, even though overexpression for chaperones can be seen. Lane 8 and 9 have no expression for VP6 (48 kDa), but chaperones' expression can be observed.
Bacteria were grown in normal Luria Broth (LB) during propagation, transformation, and induction. However, Terrific Broth (1.2% (w/v) tryptone, 2.4%
46 (w/v) yeast extract, 4% glycerol, with an additional 0.17 M K2PO4, 0.72 M K2HPO4 after autoclave) was also tried as this media increases the plasmid load in a cell.
Terrific Broth contains more nutrients than LB broth and glycerol, thus increasing the yield of bacteria per volume. The addition of potassium phosphates in the media reduced cell death by preventing the media's pH from decreasing.
All the deviations from the standard protocol still resulted in mixed results. Neither Tuner nor Origami cells gave optimal expression. Since we made these cell lines competent using our in-house one-step methods, a mutation might have occurred, which resulted in these two cell lines not expressing as they should.
No well defined expression of VP2 (102 kDa) and VP6 (48 kDa) was observed with the commercially competent cell lines Origami B and BL21 (Desselberger, 2014) (Figure 17 and Figure 18).
Figure 17: SDS-PAGE analysis of BL21_SA11_VP2. Two colonies were selected for VP2 expression. The highlighted region in lanes 2 to 5 indicate bands that cannot be observed in uninduced lanes 8 and 9. Lane 6 and 7 were used as a positive control for VP6 expression.
47 Figure 18: SDS-PAGE analysis of BL21_SA11_VP6. Two colonies were selected for VP6 expression. With the oversaturated protein concentration in each lane, no definite expression can be seen in the sample lanes nor the positive control lanes.