Materials and methods

2.2.1. E. coli BL21 competent cells preparation

Luria-Bertani (LB) Agar plates were streaked with BL21 E. coli cells (glycerol stock). The cells were grown overnight at 37 oC. After that, a single colony was placed in 10 ml LB media and left to grow overnight at 37 oC with shaking. One ml of overnight culture was used to inoculate 100 ml LB (1/100 volume) in 500 ml flask. The flask was incubated at 37 C (250 rpm), with shaking for two hours until OD600 approximately reached 0.4. The rest of the

overnight culture was stored as glycerol stock (50 % v/v glycerol) at - 80 C until required.

The cells were transferred into two pre-chilled centrifuge tubes (50 ml), and placed to chill on ice for 30 minutes. Cells were then centrifuged at 2800 rpm, for five minutes, at 4 C. Each pellet was resuspended in 12.5 ml 100 mM calcium chloride and 12.5 ml 40 mM magnesium sulfate (both pre-chilled on ice).

The resuspended cells were left to chill on ice for a further 30 minutes before they were centrifuged again at 2800 rpm, for five minutes, at 4 C. After that, the resulting pellets were resuspended each in 2.5 ml 100 mM calcium chloride and 2.5 ml 40 mM magnesium sulfate. Pre-chilled autoclaved glycerol was added to 10% total volume. The cells were placed into small fractions (250 L aliquots) before they were frozen on dry ice and stored at – 80 C until usage.

2.2.2. Genetic transformation of competent cells

50 l of competent cells were first thawed on ice. After that, 1 l of RmlA plasmid was added to the cells. The cells were incubated on ice for 30 minutes before they were exposed to a

67 heatshock for 45 seconds at 42 C (waterbath). The cells were then incubated on ice for two minutes and transferred into an incubator at 37 C with shaking for one hour.

Cells were then plated on selective media and grown overnight at 37 oC. The overnight culture was stored as glycerol stock (50 % v/v glycerol) at -80 C until required.

The plasmid encoding for RmlA from P. aeruginosa contains a sequence coding for a 6 His-tag on the N-terminus of RmlA to allow an easy purification on metal-chelating columns. The amino acid sequence for the RmlA construct is as follows:

HHHHHHGSMAMKRKGIILAGGSGTRLHPATLAISKQLLPVYDKPMIYYPLSTLMLAGI REILIISTPQDTPRFQQLLGDGSNWGLDLQYAVQPSPDGLAQAFLIGESFIGNDLSALVL GDNLYYGHDFHELLGSASQRQTGASVFAYHVLDPERYGVVEFDQGGKAISLEEKPLEP KSNYAVTGLYFYDQQVVDIARDLKPSPRGELEITDVNRAYLERGQLSVEIMGRGYAW LDTGTHDSLLEAGQFIATLENRQGLKVACPEEIAYRQKWIDAAQLEKLAAPLAKNGY GQYLKRLLTETVY

2.2.3. RmlA overexpression and purification

In order to overexpress RmlA, E. coli BL21 competent cells transformed with the plasmid were grown at 37 C in LB medium containing 100 μg/ml ampicillin until the OD600 reached 0.6 ±

0.8. Isopropyl-d-thiogalactoside (IPTG) was then added at 1 mM to induce the expression of the protein. The cell culture grew for a further 4 hours before it was centrifuged at 6000 g for 20 minutes at 4 C. Lysis buffer containing 20 mM Tris-HCl pH 8.5, 100 mM sodium chloride, 100 μg/ml hen egg-white lysozyme, was used to resuspend the cell pellet. After 30 min

68 incubation at room temperature, Ethylenediaminetetraacetic acid (EDTA) free protease cocktail inhibitor tablet (Roche) and DNase I (20 μg/ml) were added. The viscosity of the mixture was further decreased by sonication (seven cycles of one minute interrupted by one minute period on ice). The resulting lysate was centrifuged at 20,000 g for 20 minutes at 4 C to remove particulate matter.

The supernant was collected and applied, using a syringe, to a 5 ml HiTrapTM FF column (GE

Healthcare) pre-loaded with 0.1 M nickel sulfate and pre-equilibrated with buffer (20 mM Tris- HCl, pH 8.0, 500 mM sodium chloride).

The column was washed firstly with 20 column volumes of buffer containing 10 mM imidazole and followed by 30 column volumes of buffer containing 30 mM imidazole to remove non- specifically bound, histidine-rich proteins. RmlA protein was eluted with 15 column volumes of buffer containing 500 mM imidazole. Purity was assessed by SDS-PAGE (figure 2.2).

The purified RmlA was dialysed for 2 hours against 2 L of 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, and then overnight against 2 L of 50 mM Tris-HCl, pH 7.5. Dialysed, purified protein was then concentrated to 11 mg/ml for crystallisation.

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Lysate Molecular Weight

Marker (kDa) 14.4 20.1 30.0 43.0 67.0 94.0 Elution fractions (2-13) from Ni column

purification of RmlA

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 2.2: SDS-PAGE gel showing the elution fractions of RmlA during the purification

stage. The protein appeared to be pure as judged by the gel. A single band at an apparent molecular weight of 34 kDa is observed (the calculated molecular weight of RmlA based on the protein’s sequence is 33,773 Da).

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Figure 2.3: The gel filtration profile shows two peaks. The large symmetrical and sharp peak

was verified as RmlA by mass spectrometry. This column was not calibrated. However, since RmlA from P. aeruginosa was known to be a tetramer (Blankenfeldt et al., 2000a; Blankenfeldt et al., 2000b) and in this project crystallised as a tetramer it was assumed this peak corresponds to a tetramer1.

1

A subsequent purification by Magnus Alphey in the lab after I finished my thesis re-confirmed the protein elutes from gel filtration with a retention time consistent with a tetramer.

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2.2.4. The identification of the protein by Mass Spectrometry

The identity of the protein was confirmed by mass spectrometry following the in-gel digestion protocol. A band corresponding to RmlA was excised from the SDS-PAGE gel (figure 2.2) stained with Coomassie blue. The excision was as close to the boundary of the stain as possible, in a way as to minimise keratin contamination. The sample was submitted to the

Mass Spectrometry and Proteomics Facility at the University of St Andrews, where it was

reduced, alkylated and digested using the Genomic Solutions ProGest protein digestion station. Both MS and MSMS analyses were provided by the AB Sciex MALDI-TOF instrument. The sample was then processed using MASCOT, whereby mass spectra were matched against the NCBI database.

Analysis of the gel sample resulted in an unequivocal definition of the protein as RmlA, with a sequence coverage of 70% and a high score match of 1140 to Q9HU22 ( Glucose-1-phosphate thymidylyltransferase – P. aeruginosa ). Matched peptides are shown in bold red below.

1 MKRKGIILAG GSGTRLHPAT LAISKQLLPV YDKPMIYYPL STLMLAGIRE