Protein expression and purification 26

2.3.1 EcFtsZ

Escherichia coli FtsZ (EcFtsZ) was purified following the method described by Mukherjee and Lutkenhaus (Mukherjee and Lutkenhaus 1998). However, some modifications were introduced, which are detailed below.

Overproduction of EcFtsZ

To overproduce EcFtsZ, E. coli competent cells B834 (DE3) (§2.2.3) were transformed with the plasmid pETFtsZ (§ 2.2.2) which contains ftsZ open reading frame downstream of the high expression T7 promoter. 10 ml Luria-Bertani broth (LB) containing ampicillin (100 μg/ml) was inoculated with a fresh colony of the transformed B834 (DE3) and grown overnight with shaking at 37°C. The overnight culture was diluted into 1 litre of fresh LB medium with ampicillin (100 μg/ml) and grown at 37°C. At OD600 of 0.3, it was induced with 1 mM ITPG and the culture

was grown for another 3–4 hours. The culture was then chilled and harvested at 7000 RPM for 15 minutes at 4°C and washed once with cold 10 mM Tris-HCl, pH 7.9, followed by another centrifugation at 4°C. The cell pellet was kept in –20°C.

Lysis of bacterial cells

In order to observe how effective the overexpression was from the crude cell lysates, a small scale culture was lysed instead of the whole bacterial cells. Cell pellets were thawed on ice, and a small amount of the pellet was collected and resuspended in 300 µl of buffer, which contains 50 mM Tris-HCl, pH 7.9, 50 mM KCl, 1 mM ethylendiaminetetraacetic acid (EDTA) and 10% glycerol (buffer A), in a 1.5 ml eppendorf. The resuspended pellet was then lysed by sonication in a MSE Soniprep device for 30 seconds at 20% power. The sonicated extract was then clarified by centrifugation in a bench-top centrifuge for 10 minutes at 13200×g at 4°C. Samples were analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (§ 2.4.1). If the crude cell lysates gave a big band around 40 kDa on gel, which corresponds to EcFtsZ, then the lysis for the large scale of bacterial cells was performed.

From this step onward all operations were done at 4°C unless otherwise mentioned. The major part of the culture pellet (see above) was resuspended in 20 ml of buffer A, and the culture was then put on the ice and lysed by sonication in the MSE Soniprep device (70% power, 1 minute) at an interval of 1 minute. The resulting extract was then centrifuged at 10,000×g for 10 minutes to remove unbroken cells and debris and the supernatant was centrifuged afterwards at 75,600×g for 90 minutes to remove the membrane fraction. The supernatant was then collected and 30% ammonium sulfate (16.6 g/100 ml of extract) was slowly added with stirring for 20 minutes. The ammonium sulphate precipitate was then recovered after centrifugation at 20,000×g for 10 minutes and resuspended extensively in buffer A. The resuspended pellet in buffer A was dialyzed overnight against buffer A with two changes of 1 litre each to remove ammonium sulfate. Ammonium sulfate fraction of EcFtsZ was monitored by SDS–PAGE (§ 2.4.1).

Purification of EcFtsZ by ion-exchange chromatography using DEAE-Sepharose

HiPrep 16/10 DEAE FF (GE Healthcare) is a prepacked, ready to use column (bed volume, 20 ml) for anion exchange chromatography. The matrix consists of a 6% highly cross-linked spherical agarose with a mean particle size of 90 µm. The charged group in the matrix is –N+(C2H5)2H and it is stable over a pH range of 1 to 14 in the short term. EcFtsZ with an isoelectric point of 4.9 is negatively charged at pH 7.9 (after dialysis in buffer A). EcFtsZ will bind strongly to the positively charged matrix in low salt buffer. EcFtsZ can be eluted by increasing the salt gradient.

It is important to remark that rigorous cleaning was performed between purification runs as indicated by the manufacturer

(http://sbio.uct.ac.za/Sbio/documentation/DEAE_agarose.pdf). This involved passing through the column a series of solutions at a flow rate of 5 ml/min: (i) 100 ml of 2 M NaCl (which removes ionically bound proteins), (ii) 50 ml of distilled water, (iii) 100 ml of 1 M NaOH solution (which removes hydrophobically bound proteins), (iv) 50 ml of distilled water, (v) 100 ml of 70% ethanol (which removes proteins, lipoproteins and lipids tightly hydrophobically bound to the column), (vi) 50 ml of distilled water. The column was then equilibrated with 100 ml of buffer A at a flow rate of 5 ml/min.

Then the dialyzed ammonium sulfate fraction was loaded onto the DEAE-Sepharose column, which was attached to an ÄKTA purifier (Amersham Biosciences). The dialysed and concentrated fraction was washed with 100 ml of buffer A (5 bed volumes) and eluted with 200 ml of a 50 mM–1 M KCl gradient at a flow rate of 0.2 ml/min. Fractions of 2 ml were collected. During purification, proteins passing through the column were monitored by the UV absorbance at 280 nm and the peak fractions of EcFtsZ eluted at around 200–250 mM KCl.

After that, EcFtsZ fractions were also monitored by SDS–PAGE to test the purity (§ 2.4.1). The fractions that appeared as a single band with little or no contamination as judged by SDS–PAGE were pooled together and dialysed extensively in buffer A (3 changes of 3 litres each). The protein concentration was determined by the Bio-Rad reagent (§ 2.4.2.2). The protein fractions were aliquoted in 100 µl and stored at –80 °C. Purified EcFtsZ stored in this way can be stable for at least 2 years with no apparent loss of GTPase or polymerization activities.

2.3.2 EcYgfE

E. coli YgfE (EcYgfE) was produced and purified following the method described by Addinall and Johnson (Addinall et al. 2005). Using this 3 step procedure, EcYgfE was able to be purified to 99% purity allowing crystallisation. The pETDuet-1 vector (Novagen) containing the gene encoding E. coli amino-terminal six-histidine tag YgfE was kindly donated by Dr. Stephen Addinall.

Overproduction of EcYgfE

E. coli competent cells B834 (DE3) (§2.2.3) were transformed with the high expression plasmid pETDuet-1 (§ 2.2.2). 10 ml Luria-Bertani (LB) containing ampicillin (100 μg/ml) was inoculated with a fresh colony of transformed B834 (DE3) and grown overnight with shaking at 37°C. The overnight culture was diluted into 1 litre of fresh LB medium with ampicillin (100 μg/ml) and grown at 37°C with shaking until OD600reached approximately 0.5. Protein expression was

induced by the addition of ITPG to a final concentration of 0.5 mM, and then the culture was grown for another 3–4 hours. Cells were harvested by centrifugation at 6400×g at 4°C for 15 minutes, and the pellet can be kept in –20°C freezer for several days.

Lysis of bacterial cells

In order to observe how effective the overexpression was from the crude cell lysates, a small scale culture was lysed instead of the whole bacterial cells. Cell pellets were thawed on ice, and a small amount of the pellet was collected and resuspended in 300 µl of buffer A (25 mM HEPES pH 8.0, 1 M NaCl and 10 mM imidazole) in a 1.5 ml eppendorf. The resuspended pellet was then lysed by sonication in a MSE Soniprep device for 30 seconds at 20% power. The sonicated extract was then clarified by centrifugation in a bench-top centrifuge for 10 minutes at 13200×g at 4°C. Samples were analysed by SDS–PAGE (§ 2.4.1). If the crude cell lysates gave a big band around 14 kDa on gel, which corresponds to EcYgfE, then the lysis for the remainder of the culture was performed.

The large portion of the cell pellet (see above) was resuspended in buffer A. The resuspended pellet was incubated on ice for 30 minutes with 2.5 mg/ml of chicken egg white lysozyme prior to lysis by sonication in a MSE Soniprep device for 2 minutes with a minute pause interval. The sonicated extract was clarified by centrifugation at 50,000×g at 4°C for 30 minutes and applied onto a 5 ml chelating sepharose (GE Healthcare) affinity gravity flow column charged with nickel.

Purification of EcYgfE on a Ni-sepharose column by immobilised metal affinity chromatography (IMAC)

The column was washed with 50 ml (10 column volumes) of buffer containing 25 mM HEPES, pH 8.0 and 20 mM EDTA to strip any nickel and contaminants from the column. It was followed by another wash with buffer containing 25 mM HEPES, pH 8.0 without EDTA. The column was then charged with nickel by equilibrating with 50 ml of 50 mM sodium acetate buffer pH 4.0 containing 25 mg/ml nickel chloride. The column was then washed with 50 mM sodium acetate buffer, pH 4.0 to remove any unbound nickel before being finally equilibrated with buffer A.

After equilibration, the fraction was loaded onto the column and extensively washed with 50 ml of buffer A, and 50 ml of buffer B (25 mM HEPES, pH 8.0, 1 M NaCl and 100 mM imidazole). EcYgfE was finally eluted with 50 ml of buffer C (25 mM HEPES pH 8.0, 1 M NaCl and 500 mM imidazole), and 5 ml of fractions were collected.

EcYgfE was monitored during purification by SDS–PAGE (§ 2.4.1) and the protein concentration of the fractions were assayed with the Bio-Rad reagent (§ 2.4.2.2). The pure EcYgfE was finally dialysed into a buffer containing 50 mM Tris-HCl, pH 7.9, 50 mM KCl, 1 mM EDTA and 10% glycerol (the same buffer used for EcFtsZ) with 3 changes of 3 litres each. The protein was then aliquoted into 100 µl samples and stored at –80 °C until needed for further studies.