Recombinant Protein Expression Using E coli Expression Systems

Expression on all scales (unless otherwise stated) was carried out at 37oC in a shaking incubator (250rpm, New Brunswick Scientific Model G25, Innova 4430 incubator shaker, New Brunswick Scientific, St Albans, UK). For overexpression of recombinant

proteins, plasmid DNA was transformed into either DH10β or BL21 (DE3) E. coli cells,

as described in 2.1.2, using heat shock at 42oC for 45 and 30 s respectively. Otherwise, cells provided as glycerol stocks in RosettaTM2(DE3) cells were plated on LB agar containing the appropriate concentrations of antibiotic in order to isolate single colonies. All cells were harvested by centrifugation with the Sorvall© legend RT centrifuge, using the Sorvall© SH-3000 swinging bucket rotor and SS-34 fixed angle rotor (Thermo Scientific, UK).

2.3.1 Expression of RLC/MiniHMM Constructs

Original plasmids for expression of the MiniHMM fragment, human cardiac RLC and cardiac myosin binding protein C, domain C0, were provided by Dr Mark Pfuhl Kings College, London.

The antibiotics used for selection throughout were Amp and Kan at the concentrations outlined in Table 2.3. Following successful transformation of the plasmids containing the MiniHMM construct and the myosin RLC, a single colony was used to inoculate

10mL of LB broth, and the culture grown over the course of one day, before 600µL was

used to inoculate 10mL of fresh LB media. From the new culture, 500µL was added to

each litre of LB broth (for large scale expression), and cultures grown overnight at 30oC with shaking at 200rpm.

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Following overnight incubation, the temperature was increased to 37oC, and cells grown until an OD600 of ~1.00 was achieved. At this point, cells were moved to 4oC for

30min, before expression was induced overnight at 18oC, with shaking at 200rpm, with

the addition of 100mg of IPTG (dissolved in 1mL of ddH2O) per 1L of cell culture.

Cells were harvested by centrifugation at 6,000 rpm for 15min at 4oC. Supernatants were discarded and cells lysed.

2.3.1.1 Cell Lysis of RLC/MiniHMM Constructs

Following pelleting of cells, 100mL of lysis buffer was added, and cells re-suspended. Cells were kept on ice for 30min, and 10mg of lysozyme added to the mixture, with frequent stirring. To this, 1mL of Triton X-100 (25% stock) was added, giving a final detergent concentration of 0.25%, and cells kept on ice for 15min, again with frequent stirring.

Cells were frozen in liquid nitrogen for 5min before being removed and left at r.t for 10min. Cells were broken up, and 100mL of lysis buffer added with stirring at r.t for 15min, until all cells were thawed, and the mixture homogeneous. Cells were then transferred back to ice, and 500µL of DNase (2mg/mL stock) added, and cells stirred frequently on ice, until no longer viscous. Cells were diluted with a half volume of lysis buffer before being spun at 9,000rpm for 2hr at 4oC.

2.3.2 Expression of Myoglobin Mutants and cMyBP-C Domain C0

Original plasmids for the expression of sperm whale myoglobin, as well as the plasmid containing the pyrrolysyl tRNA synthetase / tRNACUA(Pyl) pair for Unnatural

amino acid incorporationwere provided by Prof Jason Chin, MRC, Cambridge.

The antibiotics used for selection were Kan and Tet at the concentration outlined in Table 2.3 for cMyBP-C and Myoglobin respectively. Following successful transformation of plasmid DNA (2.1.2) into DH10β E. coli cells, a single colony was used to inoculate 100mL of LB, supplemented with the appropriate antibiotic, and the

culture grown overnight at 37oC with shaking (200 rpm). Following overnight

incubation, 10mL of the starter culture was used to sub-culture each litre of LB. Cells

were grown until an OD600 of between 0.6 -0.8 was achieved, before protein expression

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myoglobin, and 1mM (final concentration) IPTG for the cMyBP-C domain C0. Cells were pelleted by centrifugation, supernatants discarded, and cells kept at -20oC until cell lysis by sonication.

2.3.2.1 Cell Lysis of Myoglobin Mutants and cMyBP-C Domain C0 by Sonication All cells were allowed to thaw on ice, before being re-suspended in lysis buffer. Sonication was carried out on ice, using a 30s on/off cycle for 15min at 15 amplitude microns. The suspension was clarified by centrifugation (16,000rpm, 4oC) for 30min, to precipitate cellular debris and insoluble materials, and obtain a cell free extract.

2.3.3 Expression of 6xHis TEV Protease

A glycerol stock containing RosettaTM 2(DE3) cells successfully transformed with the recombinant 6xHIS TEV protease was kindly donated by Dr Huanting Liu and Prof. James H. Naismith, University of St Andrews, Scotland, UK.

The antibiotics used for selection throughout were Cm and Amp at the concentrations outlined in Table 2.3. To 10mL of LB broth, a single colony was added, and cells grown overnight at 37oC with shaking at 200rpm, before being used to inoculate 1L of LB

broth. Cells were grown until an OD600 of 0.6 was achieved, before protein expression

was induced by the addition of IPTG at a final concentration of 0.4mM, and cells incubated with shaking overnight at 37oC. Following overnight induction, cells were harvested by centrifugation at 6,000rpm for 10min at 4oC, and the pelleted cells kept at - 70oC until ready to be lysed.

2.3.3.1 Cell Lysis of 6xHis TEV Protease by Sonication

Pelleted cells were thawed on ice, and re-suspended in 10mL of Lysis Buffer . Once homogenous, cells were sonicated on ice for 3x45s intervals, with a 1min break in between, at 15 amplitude microns, before being centrifuged at 10,000 rpm for 20min at 4oC. The supernatant was collected and further clarified by further centrifugation at 18,000 rpm for 30min at 4oC.

2.3.4 Expression of Myoglobin with the UAA pK Incorporated

The antibiotics used for selection throughout were Tet and Kan at the concentrations outlined in Table 2.3. Following successful co-transformation of the plasmids

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containing the myoglobin construct and the plasmid containing the relevant tRNA and tRNA synthetase for incorporation of the UAA pK, a single colony was used to inoculate 100mL of LB broth and this incubated with shaking (200rpm) overnight at 37oC. Following overnight incubation, the starter culture was used to seed more LB broth. Cells were grown until an OD600 of 0.6-0.8 was achieved.

At this point, the UAA pK was added at a final concentration of 2mM and 3mM for single and double mutants respectively. The UAA was dissolved in DMSO. Cells were left shaking for 30min at r.t, before L-arabinose was added at a final concentration of 0.2% to initiate induction. Cells were induced for 5hr at 37oC, before being harvested by centrifugation at 4,500 rpm for 15min at 4oC. Pelleted cells were kept at -20oC until ready to be lysed.

2.3.4.1 Cell Lysis of Myoglobin containing the UAA, pK,

Pelleted cells were allowed to thaw on ice, before 10mL of r.t BugBusterTM (MerkMillipore, Massachusetts , USA) was added, and cells re-suspended. Cells were shaken at r.t for 60min before lysed cells were pelleted by centrifugation at 10,000rpm for 20min at 4oC.

2.3.5 Expression of 14-3-3ϛ and Vps75 mutants

Agar plates from the successful transformation of the recombinant 14-3-3 ϛ and Vps75 proteins into RosettaTM 2(DE3) were kindly provided by Dr David Norman, University of Dundee, Scotland, UK. From these glycerol stocks were made for subsequent expression.

The antibiotics used for selection throughout were Amp and Cm for 14-3-3ϛ and Vps75, respectively, at the concentrations outlined in Table 2.3. To 10mL of LB broth,

a single colony was added, and cells grown overnight at 37oC with shaking at 200rpm,

before being used to inoculate 1L of LB broth.

Cells were grown until an OD600 of between 0.6 and 0.8 was achieved, before protein

expression was induced by the addition of IPTG at a final concentration of 1mM, and cells incubated with shaking for 4hr at 37oC. Following induction, cells were harvested by centrifugation at 6,000rpm for 10min at 4oC, and the pelleted cells kept at -20oC until ready to be lysed.

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2.3.5.1 Cell Lysis of 14-3-3ϛ and Vps75 Mutants by Sonication

All cells were allowed to thaw on ice, before being re-suspended in lysis buffer. Sonication was carried out on ice, using a 30s on/off cycle for 15min at 10 amplitude microns. The suspension was clarified by centrifugation (16,000rpm, 4oC) for 30min, to precipitate cellular debris and insoluble materials, and obtain a cell free extract.