Protein expression and purification 1 Expression of recombinant proteins

The Escherichia coli competent cells BL21(DE3) and C43 (DE3) (Stratagene) were used for protein expression in all cases. The latter strain contains at least one uncharacterized mutation conferring tolerance to toxic proteins, and is derived from strain C41 (DE3), which in turn derives from BL21(DE3).

Luria-Bertani (LB; 10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl) and

NaCl) were used as growth media supplemented with the appropriate antibiotic, either

ampicillin or kanamycin at a final concentration of 100 μg/ml or 35 μg/ml respectively.

For large scale protein expression 10 ml overnight starter cultures were used to inoculate 1 litre LB or TPB (plus antibiotic) cultures in 2-litre flasks, which were

incubated at 37o_{C, 180 rpm until the cells reached mid-log growth phase (OD600}

~0.6-0.8). Protein expression was then induced with 0.4 mM IPTG and cultures were

incubated overnight (~14 hours) at 25o_{C with shaking at 180 rpm. The cells were}

harvested by centrifugation at 6 krpm, 4o_{C for 15 min using a Beckman JLA 8.10000}

rotor in an Avanti J-20 XP centrifuge and stored at -80o_{C until required. The}

expression conditions of the CAS proteins in this project are summarized in table 2.1. The cloned genes not mentioned in table 2.1 were also subjected to expression trials but were either insoluble or not expressed at all.

ORF

(sso) Name Vector resistanceAntibiotic Medium Induction induction post-

growth conditions

1440 Cas3’’ pDEST14 Ampr _{TPB 0.4mM}

IPTG

25o_{C o/n}

1442 Csa2 pDEST14 Ampr _{LB 0.4mM}

IPTG 25o_{C o/n} 1441/14 42 Cas5/ Csa2 pRSFDuetHI STEV Kanr _{LB 0.4mM} IPTG 25o_{C o/n} 1443 Csa5 pET151/D- TOPO Ampr _{LB 0.4mM} IPTG 25o_{C o/n} 1437 Cas6 pET151/D- TOPO Ampr _{LB 0.4mM} IPTG 25o_{C o/n}

1986 Cmr7 pEHISTEV Kanr _{LB - 25}o_{C o/n}

1987 Cmr4 pDEST14 Ampr _{LB 0.1 mM} IPTG 25o_{C o/n} 1989 Cmr1 pDEST14 Ampr _{TPB 0.4mM} IPTG 25o_{C o/n}

Table 2.1 Expression conditions of Cas proteins from S. solfataricus

2.2.2 Purification of recombinant proteins

The general purification scheme employed in this study is the following, with

the various adjustments described for each protein in the end. Cell pellets were

resuspended at a 1:5 (w/v) ratio in affinity binding buffer (20 mM NaH2PO4/Na2HPO4

lysozyme (Sigma), 50 μg/ml DNAse I (Sigma) and protease inhibitor cocktail tablets (“Complete mini Protease Inhibitor mix EDTA-free” tablets, Roche Diagnostics, 1 tablet per 50 ml) prior to cell lysis by sonication for 12 min (6x2 min) on ice. The cell lysate

was centrifuged for 30 min at 20 krpm, 4o_{C (Beckman JA 25.50 rotor) and the}

supernatant was filtered with a sterile syringe-driven 0.45 μm filter (Milipore) prior to

purification by nickel-chelate affinity chromatography. The filtered lysate was loaded onto a 5 ml HisTrap HP Ni-sepharose column (GE Healthcare) pre-equilibrated with affinity binding buffer and the target protein was eluted over a 30 mM - 500 mM linear imidazole gradient. The protein-containing fractions were pooled together and subjected to cleavage of the six-histidine tag by the tobacco etch virus (TEV) protease

in buffer containing 20 mMNaH2PO4/Na2HPO4 pH 7.4, 500 mM NaCl, 10% glycerol,

0.5 mM EDTA, 1 mM DTT, overnight at room temperature or 4o_{C, depending on the}

protein stability. The protein sample was then applied to an equilibrated with affinity binding buffer 5 ml HisTrap HP Ni-sepharose column (GE Healthcare) in order to remove the cleaved six-histidine tag, the histidine tagged TEV protease and other contaminants. The column flow-through, containing the untagged target protein, was concentrated to an appropriate volume and then loaded onto a HiLoad 26/60 Superdex 200 gel filtration column (GE Healthcare) equilibrated with the appropriate gel filtration buffer (depending on the protein and the subsequent purification step) from which the protein eluted as a single monodispersed peak. The gel filtration buffers used consisted of 20 mM MES pH 6 or 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.5 mM DTT, 10% glycerol. All chromatographic purification steps were performed on a BioLogic DuoFlow chromatography system (Bio-Rad) and an AKTA Xpress automatic protein purification system. The efficiency of the procedure and the purity of the sample at each purification step was confirmed by SDS-PAGE electrophoresis on 4-12% NuPage gels (Invitrogen). The identity of the target protein was confirmed by MALDI-TOF and ESI mass spectrometry. Purified protein samples were pooled together, concentrated to an appropriate concentration with Vivaspin concentrators (Sartorius Stedim Biotech GmbH), flash-freezed in liquid nitrogen and

stored at -80o_{C. The protein-containing samples were kept on ice at all times, unless}

otherwise stated.

Cas3’ (Sso1440) (both wild-type and mutant versions) was purified as

described above and stored in a final buffer containing 20 mM MES pH 6, 500 mM NaCl, 1 mM EDTA, 0.5 mM DTT, 10% glycerol. All purification procedures were carried out on ice due to partial protein degradation, unless otherwise stated.

Csa2 (Sso1442) was purified as described above with the addition of a third purification step, a 5 ml HiTrap Heparin HP column pre-equilibrated with a 20 mM MES pH 6, 50 mM NaCl, 1 mM EDTA, 0.5 mM DTT, 10% glycerol buffer, from which the protein eluted with a 50 mM - 1 M NaCl linear gradient. The six-histidine tag was uncleavable.

For the purification of the Csa2/Cas5 protein complex all procedures were carried out at room temperature and in the absence of DNAse to prevent carry-over

contamination. The soluble fraction was incubated at 65o_{C for 20 min to precipitate}

most of the E. coli contaminant proteins and centrifuged at 40 krpm in a Beckman

Coulter Optima L-90K Ultracentrifuge with a Beckman 70Ti rotor prior to metal-chelate affinity purification. The six-histidine tag was not cleaved and a third purification step on a 5 ml HiTrap Heparin HP column was employed as described for Csa2. In the case of the Csa2/Cas5 complex the purification through a heparin column enabled also the separation of the biologically relevant complex dimer from the excess of expressed Csa2 subunits.

Cas6 (Sso2004) was purified by Dr Shirley Graham as described above and stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 % glycerol.

Cmr4 (Sso1987) and Cmr1 (Sso1989) were purified as described above, with the addition of 0.5% Triton X-100 in the lysis buffer.

Protein concentrations were calculated from the sample absorbance at 280

nM, measured on a Varian Cary 50Bio UV-Visible spectrophotometer, and the theoretical molar extinction coefficient obtained from the ExPASy ProtParam analysis of the protein sequence, using Beer Lamber’s law. In the case of Csa2 (Sso1442) which lacks tryptophan residues, the Bradford reagent (Bio-Rad) was also used to calculate protein concentrations as per the manufacturer’s instructions using a BSA standard curve.