PREÁMBULO

IPTG induction and Auto-induction

The small scale IPTG induction method was used to test solubility. The S. aureus

mevalonate kinase plasmid was firstly transformed into E. coli BL21 (DE3) cells

(Novagen) and plated onto Ampicillin agar plates. Single colonies were grown in 10

ml LB medium at 37 ℃ for 4 hours, grown to an OD600 of 0.6–0.8 at 37 ℃,

and 16 ℃, respectively. Protein over-expression was achieved by induction with 1.0

mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 6 hours.

The auto-induction method was employed to test the expression and solubility (Studier 2005). 1ml of a 10 ml overnight culture was used to inoculate 50 ml of LB

and the cells were harvested, after overnight incubation at 37 ℃, at 2000 g and

re-suspended in 20 ml PBS. This was then used to inoculate auto-induction medium for over-night expression of protein, as detailed by Studier (Studier 2005). 500 ml of auto-induction growth medium in a 2L baffled flask, supplemented with 100 µg/ml

ampicillin, was inoculated with 5 ml re-suspended cells and grown at 37 ℃, using

shaking at 300 r.p.m for 4 hours to start bacterial growth. The temperature was then

lowered to 16 ℃ and left for 48 hours to reach saturation (OD600 ~ 2.6). The results

indicate that more soluble S. aureus MK resulting using the auto-induction method

(FIGURE 4.5). Large scale (4 L) expression using auto induction was then performed to produce the target protein. Induced cells were harvested by centrifugation at 10500 x g for 15 minutes (Beckman Avanti J20-XP, JL8.100 rotor). The cells were

re-suspended in PBS to remove any excess growth media and stored at -80 ℃ until

FIGURE 4.5: Coomassie Blue stained SDS-PAGE gels of S. aureus PFK. [A.1] protein marker. [A.2] crudes flow through of IPTG induction at 16 ℃.[A.3] first elution of IPTG induction at 16 ℃. [A.4]crudes flow through of IPTG induction at 25 ℃. [A.5] first elution of IPTG induction at 25 ℃. [B.1] crudes flow through of auto induction at 16 ℃. [B.2] first elution from auto induction at 16 ℃. [B.3] first elution from auto induction at 25 ℃. [B.4] first elution from auto induction at 25 ℃.

Purification

A standard purification protocol was applied as described in chapter II. The buffer system for MK purification is slightly different from PFK protein. 50 mM phosphate

buffer (11.25 mM NaH2PO4, 38.75 mM Na2HPO4) replaced the 50 mM Tris-HCl in S.

aureus MK purification approach. The induced cells were re-suspended in binding

buffer at room temperature for 30 minutes. The cells were disrupted by sonication for 8 cycles of 45 seconds, interrupted by 1 minute periods on ice after addition of 20µg/ml DNAse I and one tablet of protease inhibitor (ROCHE). To ensure that the

DNA was digested by DNAse I, the mixture was kept at 4℃ for another 20 minutes.

After addition of 2 mM EDTA, the mixture was centrifuged for 45 minutes at 105000

(Millipore) and collected for further purification.

The supernatant containing mevalonate kinase protein was applied to a charged Histrap Nickel Sepharose, high performance column (Amersham Biosciences), which had been equilibrated already with binding buffer. Unbound and weakly bound proteins were removed by extensive washing with washing buffer. Essentially pure mevalonate kinase protein was eluted and collected with elution buffer. Eluted protein was incubated with 1 ml of 2 mg/ml tobacco etch virus protease (TEV) (approximate ratio 20:1 protein: protease) to remove the polyhistidine tag at room temperature

(25 ℃). The MK protein and TEV mixture was dialyzed against 500 mM NaCl, 2 mM

DTT, 50 mM phosphate buffer, pH 8.0 and 10% glycerol for 6 hours to remove imidazole. The progress of cleavage was checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (FIGURE 4.6).

FIGURE 4.6: Coomassie Blue stained SDS-PAGE gels of S. aureus MK. [A.1] protein marker. [A.2] crudes flow through. [A.3] first wash through. [A.4]first elution. [A.5] second elution. [B.1]protein marker. [B.2]before tev cleavage. [B.3] 2 hours after addition of tev. [B.4]6 hours after addition of tev. [B.5] elution from second nickel column.

The protein-TEV mixture was passed through a 0.22 μM syringe filter membrane (Millipore) and a second Ni-NTA column to remove any non-cleaved protein, tag and protease (Liu and Naismith 2005). Then the flow through was concentrated to approximately 5 mL before further purification by size exclusion gel filtration. Some

precipitant was removed by centrifugation at 75000 x g for 10 min (4℃, Beckman

Avanti J20-XP JA25.50 rotor). Size exclusion gel filtration was applied on a Superdex™ 200 size exclusion chromatography column (Amersham Biosciences) After the gel-filtration step proteins were judged to be pure by Coomassie™ Blue-stained gels and their integrity was confirmed by mass spectrometry (University

of St Andrews) (FIGURE 4.7). In total it was estimated 20 mg pure S. aureus

mevalonate kinase native protein was obtained from 4 L of cell culture. According to the results of the Pre-Crystallization Test (PCT), the protein was then concentrated to 10 mg/ml and 5 mg/ml respectively prior to crystallization. Some of the protein was flash-frozen in liquid nitrogen for long term storage. The buffers used in each step were summarized in TABLE 4.1

Binding buffer 500 mM NaCl, 2 mM DTT, 50 mM phosphate buffer, pH 8.0, (11.25 mM NaH2PO4, 38.75 mM Na2HPO4), 10 mM imidazole

Wash buffer 500 mM NaCl, 2 mM DTT, 50 mM phosphate buffer, pH 8.0, (11.25 mM NaH2PO4, 38.75 mM Na2HPO4), 35 mM imidazole

Elution buffer 500 mM NaCl, 2 mM DTT, 50 mM phosphate buffer, pH 8.0, (11.25 mM NaH2PO4, 38.75 mM Na2HPO4), 500 mM imidazole

Dialysis buffer

500 mM NaCl, 2 mM DTT, 50 mM phosphate, pH 8.0, (11.25 mM NaH2PO4, 38.75 mM Na2HPO4), 500 mM imidazole, 10% glycerol.

1mM EDTA Gel-filtration buffer

(Crystallization buffer)

150 mM NaCl, 50 mM phosphate buffer (11.25 mM NaH2PO4,

38.75 mM Na2HPO4), pH 8.0

FIGURE 4.7: Gel-filtration results and mass spectrometry analysis of S. aureus mevalonate kinase. [A] selected fraction of gel-filtration elution. [B] mass spectrometry output of the protein.