Validación del procedimiento a través del Método de Expertos

To confirm the expression and identification of MraY mutants, two methods were explored. An empty pET52b vector was transformed in E. coli C43 cells. The corresponding membranes were isolated in a similar manner to the MraY mutants. These empty membranes contained only endogenous membrane proteins. Empty membranes were then diluted to 23mg/mL and 13mg/mL and compared to MraY mutants using the continuous fluorescence assay. In all three cases, the fluorescence emission spectrum showed that mutants F288A, F288L and E287A were overexpressed relative to the empty membranes (Figure 3.11). The reaction of F288L resulted in lower fluorescence relative to the other mutants; this is presumably due to the lower protein concentration.

The activity of F288A, E287A and F288L MraY mutants were further confirmed by the addition of UMP. Le Chateliers principle states that any change in a system at equilibrium will result in a shift of the equilibrium to counteract the change210. Given that UMP is another product of the reversible MraY reaction, the addition of UMP

101 was expected to shift the equilibrium towards the formation of substrate. To the negative control, 15μL of UMP (final concentration 83μg/mL) was added to the MraY reaction. The addition of UMP resulted in a decrease in fluorescence indicative that dansyl-Lipid I is being converted back to UDP-MurNAc-(Nε-Dns)pentapeptide and exported out of the membrane.

a)

b)

UMP

102

Figure 3.11: Overexpression of MraY mutants relative to “empty” membranes.

a) E287A MraY at a final concentration of 2mg/mL b) F288A MraY at a final concentration of 1mg/mL c) F288L MraY at a final concentration of 2mg/mL. Given that UMP is another product of the reversible MraY reaction, the addition of UMP caused a decrease in fluorescence indicative that the equilibrium has shifted towards the reformation and exportation of the substrate out of the membrane.

Another approach utilised to confirm the expression and identification of MraY mutants was Western blotting. There are very few examples of successful western blots in the characterisation of MraY. Lloyd et al characterised C-His6-tagged MraY

in 2004 upon extraction from the membrane71. Ma et al (2011) characterised C-His6-

tagged MraY produced from cell-free expression techniques in the presence of artificial lipids and detergents211. In both studies, the MraY band ran lower than expected at 32kDa.

A Western blot was performed on membrane-bound MraY mutants using Strep-Tactin Horse Radish Peroxidase (HRP) conjugate antibodies. Unfortunately, no bands were observed for MraY mutants. This could be due to the use of membrane bound MraY. The extensive lipid environment may have hindered recognition by the Strep-Tactin

c)

103 HRP conjugated antibody. In addition, due the hydrophobic nature of membrane proteins like MraY, aggregation may have occurred upon denaturation212.

Given the possibility that aggregation may have occurred preventing the protein from traveling down the SDS-gel and transferring correctly to a PVDF membrane, a Dot blot was performed. A dot blot is often used clinically in the detection of sexually transmitted diseases such as chlamydia and in the detection of antidiacyltrehalose antibodies in tuberculous patients213. A dot blot is a simple and quick technique for detecting and identifying proteins in the presence of antibodies. Unlike a Western blot, a dot blot cannot distinguish proteins by size; it is only able to confirm the presence of a tagged protein upon binding to an antibody214.

To a nitrocellulose membrane paper, 5 and 20μL of membrane-bound MraY mutants (13 mg/mL) were spotted directly and allowed to air dry. To prevent nonspecific binding, the membrane was treated with 5% Bovine Serum Albumin (BSA) in PBS for 1 hour at 4°C. Following three consecutive washes with PBS-Tween buffer, the membrane was treated with 20μL of diluted Strep-Tactin HRP conjugate in 10mL of PBS-Tween buffer for 1 hour at 25°C with gentle shaking. The membrane was washed twice with PBS-Tween and PBS. The membrane was finally treated with Novex® Electrochemiluminescence (ECL) reagents which contained an electrochemiluminescence substrate used for immunodetection of HRP (Figure 3.12). The dot blot was subsequently exposed to film and developed using an AGFA Curix 60 processor.

104

Figure 3.12: Chemiluminescence reaction of Strep-Tactin HRP conjugate

Incubation with ECL reagents for 30 seconds followed by 15 seconds exposure to film produced Figure 3.13a. ECL was able to detect the Strep-Tactin HRP conjugate antibody confirming the presence of Strep-tagged MraY in these samples. A small amount of nonspecific binding was observed in the negative control. Nonspecific binding commonly results from insufficiently blocking with BSA or an excessive concentration of the antibody215.

Incubation with ECL reagents for 2 minutes followed by 60 seconds exposure to film produced Figure 3.13b. Prolonged incubation with ECL lead to the formation of halo like circles in the dot blot. These halos are often called ghost bands and are a result of high protein concentrations and long incubations times with ECL reagents216. A high concentration of protein results in a high concentration of the bound Strep-Tactin HRP conjugate. The addition of the ECL reagents resulted in a fast conversion of substrate to product. This led to a decrease in substrate concentration and therefore a decrease in the electrochemiluminescence reaction overtime. Because this reaction was not exposed to film quickly, the electrochemiluminescence reaction could not be accurately detected.

105

Figure 3.13: Dot Blot for MraY mutants.

a) 30 seconds incubation with ECL followed by 15 seconds exposure to film b) 2 minutes incubation with ECL followed by 1 minute film exposure. Excess ECL exposure time led to a lack of HRP substrate upon film exposure.