X Binding Buffer 4 X Elute Buffer X Charge Buffer 40mM imidazole

4M NaCl 160mM Tris-HCl pH7.9 4M imidazole 2M NaCl 160mM Tris-HCl pH7.9 400mM N1S0 4

4 X Wash Buffer 4 X Strip Buffer

960mM imidazole 4M NaCl 320mM Tris-HCl pH7.9 400mMEDTA 2M NaCl 80mM Tris-HCl pH7.9

Table 2.10 Solutions for Nickel-binding Chromatography

All solutions were diluted with sterile water to 1 x just prior to use.

In general, the manufacturer's protocol was followed. Only the W ash buffer differed from that recommended by the manufacturers. Rather than the 4M NaCl contained in the original 8 x Wash, a 4 x buffer containing 4M NaCl was used. The concentrations of other components of the W ash buffer were doubled accordingly such that the final 1 x W ash B uffer consisted of 240mM imidazole, IM NaCl and SOmM Tris-HCl. This relative doubling of NaCl concentration was found to remove non-specifically bound material from the column to leave only nickel-bound protein.

Protocol

The procedure was perform ed at room tem perature, as per the m anufacturer's instructions. The 2.5ml column matrix (suspended in 20% ethanol for storage at 4°C) was poured into a 10 x 1cm plastic column. When settled, the matrix was washed with 15ml deionised water, and then charged with 12.5ml Charge Buffer. The Binding Buffer (7.5ml) was applied; it equilibrates the system and imidazole binds the Ni ligands. After Binding Buffer had drained to the top of the column, the prepared extract was loaded and allowed to run through; any molecules present able to bind Ni and displace imidazole will do so.

The volume of bacterial cell extract loaded onto the column depended on concentration of nickel-binding protein present. The protein content of the soluble cell extract of the bacterial strain was assessed by SDS-PAGE and an estimate of protein concentration made. For the soluble cell extract prepared from 3 litres of overnight bacterial culture and dissolved in 15-

20ml of T/G 7.5 buffer (see Table 2.8), the 2.5ml matrix was sufficient.

The column was washed with 25ml Binding Buffer to occupy vacant Ni residues and to equilibrate the matrix, and the Wash Buffer (15ml) was passed through to remove any un­ bound material remaining. In order to remove bound material, the Elute Buffer (5ml), containing a high concentration of imidazole, was used, and 0.5ml fractions collected. Strip Buffer was applied to remove any remaining material. At each stage, fractions were collected and analysed by SDS-PAGE. W ith practice, it was possible to collect all the protein removed by the Elute Buffer in one 3-5ml fraction.

The results of this work, and slight adaptations to this method, are shown in Chapter 4

2.3

Protein Assessment

2.3.1

Protein Sequencing

2.3.1.1 S D S -P A G E

The SDS-polyacrylamide protein gel was prepared and run as described in Section 2.2.2. In order that polymerisation be complete, the separating gel was left to stand overnight and wrapped in cling film to prevent drying out. Otherwise, reactive species within the gel could interfere with the proteins being separated. The stacking gel was also left to stand for 4 hours before use. The gel was loaded with denatured samples and run for 45 minutes at 40mA.

2.3 .1 .2 Protein B lotting

The SD S-PA G E gel was blotted by the total im m ersion system. B lotting was accomplished using a BioRad system, consisting of two perspex plates perforated with 1cm diameter holes between which the sandwich is assembled. Assembly was carried out in a large shallow tray under 1 x CAPS buffer (pH l 1).

The protein gel was placed adjacent to a PVDF (polyvinylidene difluoride) blotting

m em brane betw een layers of W hatm an 3MM paper as shown in F ig u re 2.1. The

membrane was wetted in neat methanol and then equilibrated in CAPS buffer prior to use. Bubbles were removed from between the layers by rolling a glass rod over them and then fastening the two electrode plates together.

The sandwich apparatus was run in 2.5 litres of 1 x CAPS buffer at 30V for 80 minutes at 4°C, during which time the protein migrated onto the membrane.

#»#>##)#><## #)(# # # # # # # # # # ' ®. # . # # # # # # #

cathode perspex plate sponge filter paper SDS-PAGE gel PVDF membrane filter paper sponge

anode perspex plate

F igure 2.1 Blotting Apparatus

The 'sandwich' used for blotting of proteins onto a PVDF membrane. Assembly was carried out under 1 x CAPS buffer at all times.

2.3.1.3 PVDF M em brane Staining and Drying

After blotting, the membrane was withdrawn from the sandwich and immersed in amido black stain for 10 minutes with constant agitation. It was then transferred to de-stain (7% v/v acetic acid, 10% v/v methanol) for 20 minutes, with regular changes of de-stain solution. After drying in a 37°C oven for 30 minutes, the appropriate protein band was excised from the membrane with a sterile scalpel blade.

N-terminal protein sequencing was carried out by Dr. Lawrence Hunt (University of Southampton).

2.3.2

Spectroscopic Analysis of Tetrapvrrole Biosynthesis

2.3.2.1 Propogation and H arvesting o f Bacteria

Bacteria were grown in M9 medium containing 5% LB, lOmg/1 ALA, and lOOmg/1 ampicillin overnight at 37°C with constant shaking. If appropriate, cobalt was also added to a final concentration of 5pM after 2 hours growth. The bacterial pellet was harvested by centrifugation at 12,500 x g for 10 minutes at 4°C in a Heraeus Sepatech centrifuge (14 x 50ml rotor size, Heraeus HFA 18.50) and then resuspended in 2ml lOmM Tris-HCl (pH 7.5).

2.3 .2 .2 Isolation o f Non-Protein M aterial from Bacteria

The bacterial soluble cell extract was obtained by sonication in an ice/water bath ( 4 x 1 minute bursts at 10 microns), and centrifugation for 5 minutes at 10,000 x g in a micro­ centrifuge. The supernatant was mixed on ice for 1 minute with 1ml of resuspended DEAL Sephacel resin. After washing with 3ml lOmM Tris-HCl (pH7.5), remaining material was eluted with 2ml lOmM Tris-HCl (pH7.5) + IM NaCl.

2.3.2.3 S p e c tr o sc o p y

The absorbance spectra of sirohaem and its immediate precursors are well characterised. To assess the ability of bacterial enzymes to produce these compounds, the uv-visible spectrum o f the final material was obtained with a Hewlett-Packard 452A Diode Array Spectrophotometer (pathlength 1cm) between 300 and SOOnm using Hewlett-Packard UV- Visible ChemStation software.