Protein expression and purification

MMT6 Buffer A 

20 mM HEPES pH 7.5, 10% (v/v) glycerol, 800 mM Nacl, 20 mM Imidazole, 2 mM β-mercaptoethanol and ETDA free protease inhibitors 

MMT6 Buffer 

B 20 mM HEPES pH 7.5, 10% (v/v) glycerol, 150 mM Nacl, 20 mM Imidazole, 2 mM β-mercaptoethanol, and ETDA free protease inhibitors 

MMT6 Buffer B1 (High Imidazole) 

20 mM HEPES pH 7.5, 10% (v/v) glycerol, 150 mM Nacl, 500 mM Imidazole, 2 mM β-mercaptoethanol, and ETDA free protease inhibitors 

MMT6 Buffer B2 (High NaCl) 

20 mM HEPES pH 7.5, 10% (v/v) glycerol, 800 mM Nacl, 20 mM Imidazole, 2 mM β-mercaptoethanol and ETDA free protease inhibitors 

MMT6 Buffer C 

20 mM HEPES pH 7.5, 10% (v/v) glycerol, 300 mM Nacl, 2 mM β-mercaptoethanol and ETDA free protease inhibitors 

1x 5ml HiTrap™ FF Ni²⁺ column 

1x 5ml Hitrap™ SP Sepharose HP column 

1x 5ml HiTrap™ Q Sepharose HP column 

His-tagged Tobacco Etch Virus (TEV) protease 

1m=x 1ml mMnoQ column 

AKTA FPLC 

1. Codon Plus Escherichia coli cells expressing MMT6 were recovered from glycerols. These were over expressed in Auto Induction Media (AIM) containing kanamycin by incubating overnight at 37℃. 
2. The Cells were harvested by centrifugation at 4℃ and resuspended in MMT6 Buffer A, and lysed by freeze thawing and cell disruption by nitrogen decompression. 
3. Lysates were then centrifuged for an hour at 4℃ and the supernatant was passed over a HiTrap™ FF Ni²⁺ column (2 ml/min). 
4. The column was then washed with Buffer B. The Protein was then eluted by stepping off using Buffer B1. 
5. Eluted fractions were then passed through a HiTrap™ SP Sepharose HP column (2 ml/min) in Buffer B to remove proteolysed B-subunits and the flow through collected. Waste protein was stepped off with Buffer B2. 
6. The flowthrough was then loaded on to a HiTrap™ Q HP column 2 ml/min) in MMT6 Buffer B. The protein was then eluted by stepping off with MMT6 Buffer B2. Run SDS page gel. 
7. Eluted fractions were pooled and protein concentration was determined via Bradford Assay. This was then incubated overnight at 4℃ with His-tagged Tobacco Etch Virus (TEV) protease in a 1: 50 weight for weight ratio (TEV protease: MMT6). 
8. The protein was then passed over a second subtractive Ni2+ affinity column in Buffer B to remove uncleaved protein, his tags and his tagged protease. The flow was collected and the waste stepped off with MMT6 Buffer 1. The collected flow through was then dialysed overnight at 4℃ into MMT6 Buffer C. Enzyme was then recovered, activity and concentration determined by relaxation and Bradford assay respectively. Enzyme was then aliquoted, flash frozen in liquid nitrogen and stored at -80℃ 

Removal of Nucleotide component in Protein Prep 

One of the final flowthrough fractions from the MMT6 protein purification was recovered and thawed on ice. Fractions selected had been tested positive for activity and the presence of the nucleotide component. A Mono Q column was equilibrated in MMT6 Buffer B with the flow rate over it was adjusted to minimise the back pressure. The thawed protein was then passed through the column and washed in MMT6 Buffer B. MMT6 Buffer B2 was then used to increase the salt concentration in the column over the gradient: 150-800mM NaCl over 25ml at 0.5 ml/minute. All flow-through fractions were collected and 50 µl samples were taken from each fraction for analysis by SDS-PAGE. The fractions identified with containing protein were concentrated and tested for activity using relaxation and decatenation Assays. Flowthrough fractions containing the nucleotide component (DNAϝ) were identified using spectrophotometer and confirmed by DNA gel electrophoresis.