Purification of CPR5 protein variants

Sample preparation

Bacteria containing transgenic plasmids were grown in 5 mL LB (Section 2.6.1) containing appropriate antibiotic(s) and incubated overnight at 37°C at 200 x rpm. Having achieved an optical density of 0.5 – 0.6, 2.5 mL, this culture was added into two flasks, each containing 100 mL of LB and appropriate antibiotic(s), and then incubated on 37°C at 200 x rpm. After saving 1.0 mL from each flask as the un-induced control, the rest of the culture was induced by IPTG (1 mM) and incubated at 37°C and 200 x rpm. After 4 hrs, 30 mL from each flask (the induced culture) was taken out and the rest was kept shaking overnight (O/N) under the same conditions as mentioned above, in order to compare protein expression and stability. Cells from each sample (un-induced, induced for 4 hours and overnight) were collected in the form of pellets by spinning at 4000 x rpm at 4°C for 10 minutes and then frozen at -800C. Pellets of cells from each sample were thawed and re-suspended in 3.0 mL and 1.5 mL of lysis buffer (Section 2.9.5.1). 250 uL from both of these samples (3.0 mL and 1.5 mL tubes) were divided into 1.7 mL tubes and then the cells were lysed by sonication (20%, 100J). The lysed cells were spun at 13,200 rpm for 1 minute, and a supernatant containing the soluble protein was transferred into new tubes, whereas the left-over cell pellet was dissolved in 8.0 M Urea (Section 2.9.5.5). The expression level of each construct in the soluble fraction of protein was checked and

41

compared to the insoluble fraction of the protein on 12% SDS-PAGE gel (Section 2.9.7) before the purification of the protein through His-column or ion-exchange chromatography.

Resin (sepharose beads) preparation

150 μL of chelating Sepharose (GE healthcare, Life Sciences) was added into a 1.7 mL tube and spun for 7-10 sec. The resin was washed with 70% ethanol (three times) as well as with double distilled water (ddH2O) five times, and the supernatant was discarded each time in order to remove the residual ethanol from the resin. 75 μL of the washed resin was added into the tube containing Ni+ metal ions (or Co+, Fe+, Zn+, Mg+; Section 2.9.5.6) and was subsequently washed with double distilled water (ddH2O) five times. Resin was equilibrated with lysis buffer (the same buffer with which the cells were lysed) five times.

Purification using His-tag

Cell lysates (supernatants and pellets prepared in Section 2.9.1) were mixed into the tubes containing sepharose beads (resin) pre-charged with selective metal ions (Section 2.9.5.6) and the mixture was incubated for 1.5 hrs at 4°C and continuously swirled. After 1.5 hours of shaking, the tubes were spun for 30 seconds at full speed and the supernatants were transferred to fresh tubes. Resin containing proteins was washed with 1 mL of elution buffer containing low levels of imidazole (Section 2.9.5.3) and the supernatants were saved. To avoid any protein degradation, protein inhibitors were added to all the supernatants obtained as a result of each washing and kept on ice during the purification process. The resin was washed with elution buffer containing 100 mM of imidazole in order to remove non-specific (bacterial) proteins weakly bounded onto the resins, and the supernatants were transferred to new tubes. In order to elute CPR5 His-tagged proteins, the resin was washed with elution buffers (Section 2.9.5.6) containing 0.5 M, 1.0 M and 1.5 M imidazole in a gradient manner, and all the supernatants were transferred to fresh tubes. All the supernatants isolated from un-induced and empty vector samples were migrated onto SD-PAGE gels at 200 V for 55 minutes. The gels were subsequently stained in Coomassie blue staining for 45 minutes. Gels were de-stained in de- staining buffer and imaged by scanning.

Purification using ion-exchange chromatography

In order to purify the protein using ion-exchange chromatography (without His-tag), the isoelectric point (pI) of each construct (Untagged version) was calculated using ProtParam

42

(http://expasy.org/). Since most of the constructs displayed an isoelectric value of 6.92-7.58, both anion and cation exchange chromatography were used in order to purify different untagged versions. Based on the pI value of each construct, two types of lysis, loading and elution buffers (Section 2.9.6),such as, sodium acetate (pH 5.0) for anion chromatography and Tris-HCl (pH 8.0) for cation chromatography, were prepared. Samples were prepared and processed for purification in a similar fashion as prepared for the His-tagged version in Section 2.9.3.

List and recipes of buffers used for Ni+_{-column based purifications}

2.9.5.1Lysis buffer Chemical 250mL 500mL 50mM K2HPO4 (pH=7.5) 2.1773g 4.3545 g 0.25M NaCl 6.13g 12.26 g 10% (v/v) glycerol 25mL 50 mL 0.5 mM TCEP 35.827mg 71.655 mg 10mM Imidazole 170.2mg 340.4 mg

The final pH was adjusted between 7.5 8.0 using KCl. The buffer was filtered and stored without autoclaving. 2.9.5.2Loading buffer 50 mM K2HPO4 (pH=7.5) 2.1773 g 4.3545 g 0.5 M NaCl 12.26 g 24.52 g 0.5 mM TCEP 35.827 mg 71.655 mg 20 mM Imidazole 340.4 mg 680.8 mg

The final pH was adjusted between 7.5 8.0 using KCl. The buffer was filtered and stored without autoclaving. 2.9.5.3Elution buffer Chemical 250mL 500mL 50 mM K2HPO4 (pH=7.5) 2.1773 g 4.3545 g 0.5 M NaCl 12.26 g 24.52 g 0.5 mM TCEP 35.827 mg 71.655 mg 500 mM Imidazole 8.51 g 17.02 g

43

The final pH should be 7.5 or 8.0. The buffer was filtered and stored without autoclaving. 2.9.5.410x PBS buffer

KH2PO4 0.6 g 1.2 g

NaCl 20.0 g 40.0g

KCl 0.5 g 1.0 g

Na2PO4.7H2O / Na2PO4 6.7 g/3.6 g 13.4 g/7.2 g

All of the above mentioned chemicals were dissolved in ddH2O and stored after sterilizing. Notes

1. K2HPO4 is a strong base so adjust pH first with HCl by diluting it to 100 folds 2. Imidazole is light sensitive, so keep out of light by wrapping in aluminium foil 3. Don’t forget to wash the filter with ddH2O after filtering each buffer

4. The pH of all buffers should be the same i.e. 7.5 or 8.0 2.9.5.5Preparation of 8 M urea

8 M Urea 14.41 g 15 mL of distilled water

Distilled water was added to make up the final volume to 30 mL. It was not filtered or sterilized and was stored at room temperature.

2.9.5.6Preparation of metal ions used in chromatography

Ion Amount (mg) ddH2O (mL) Ni+ _(NiCl 2) 200 1.0 Co+ _{(CoCl2 ) 200 1.0} Fe+ _(FeCl 3 ) 200 1.0 Zn+ (ZnCl2 ) 200 1.0 Mg+ (MgCl2) 200 1.0

Selected ions were completely dissolved and filtered.

List and Recipes of buffers used for ion-exchange purifications

A. 10 mM Tris-HCl (pH= 8.0 with HCl); dissolved 20 mL of 50 mM Tris-HCl buffer (which also contained 0.05 mM TCEP) in 80 mL ddH2O

B. 10 mM Tris-HCl + 1M NaCl (pH= 8.0); dissolved 20 mL of 50 mM Tris-HCl buffer in 80 mL ddH2O in addition to 5.844 g of NaCl

C. 10 mM NaOAc (pH= 5.0 with acetic acid)

44 Preparation of 12% acrylamide gels

2.9.7.1Preparation of resolving or separating gel

Monomers 5mL 10mL 15ml 20ml ddH2O 2.18 mL 4.345 mL 6.53 mL 8.69 mL 1.5 M Tris-HCl 1.25 mL 2.5 mL 3.75 mL 5.0 mL 20 % SDS 25 μL 50.0 μL 75.0 μL 100.0 μL 40 % Acrylamide 1.5 mL 3.0 mL 4.5 mL 6.0 mL 10 % APS 50.0 μL 100.0 μL 150.0 μL 200.0 μL TEMED 2.5 μL 5.0 μL 7.5 μL 10.0 μL

2.9.7.2Preparation of stacking gel

Monomers 5mL 10mL 15ml 20ml ddH2O 3.12 mL 6.24 mL 9.36 mL 12.48 mL 1.5 M Tris-HCl 1.25 mL 2.5 mL 3.75 mL 5.0 mL 20 % SDS 25.0 μL 50.0 μL 75.0 μL 100.0 μL 40 % Acrylamide 0.55 mL 1.1 mL 1.65 mL 2.2 mL 10 % APS 50.0 μL 100.0 μL 150.0 μL 200.0 μL TEMED 5.0 μL 10.0 μL 15.0 μL 20.0 μL WARNING:

 De-gas the mixture after adding 10% APS  Immediately cast gel after adding TEMED  Un-polymerized acrylamide is extremely toxic  Wear gloves and never pipette by mouth