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Construction of expression vector and expression:
The pSESAME-Cre expression vector was constructed by inserting a Cre-encoding fragment into pSESAME via AvrII and NheI restriction sites using standard cloning methods. pSESAME encodes a fusion protein consisting of a histidine-tag, TAT-domain, NLS sequence and Cre, abbreviated HTNCre. For expression of HTNCre the pSESAME-Cre was transformed into TUNER (DE3) pLacI and used to prepare a glycerol stock.
- An over-night culture was inoculated using a pipet tip coated with transformed bacteria from the glycerol stock. The over-night culture consisted of LB media supplemented with 0.5% glucose [v/v] and carbenicillin at a final concentration of 50 μg/mL and was allowed to grow at 37°C for 16 hours.
- Next day the densely grown over-night culture was used to inoculate the expression culture at a ratio of 1 to 40 and was put in an incubator at 37°C. Expression culture consisted of TB media supplemented with 0.5% glucose [v/v] and ampicillin at a final concentration of 100 μg/mL.
- At an OD595 of 1.5 the expression culture was induced with 0.5 mM IPTG for 1 h.
- Subsequently bacteria were collected by centrifugation at 5000 rpm for 10 minutes in a SLA3000 rotor.
- Bacteria pellets were stored at minus 20°C until purification.
Purification of cell-permeable protein:
- Frozen bacteria pellets were resuspended in 10 mL lysis buffer per liter flask culture for 15 minutes at room temperature.
- Suspension was then incubated with 1 mg/mL lysozyme for additional 15 minutes while mixing at room temperature.
- 25 U/mL benzonase was added afterwards and incubated while mixing for 15 minutes at room temperature.
- After sonification on ice for 1.5 min with 0.5 s pulses at 45% of the power, 1 mL cold tartaric salt buffer (TSB) per mL suspension was carefully added while mixing and incubated for 5 min on ice. SDS-PAGE sample of lysate fraction (L) was taken.
- Cleared lysate was obtained by centrifugation at 4°C for 30 min at 30,000g. SDS-PAGE samples of soluble (S) and insoluble fractions (I) were taken.
- The supernatant was transferred into fresh 50 mL falcon tubes and was then gently mixed for 1 h at 4°C with 2 mL of 50% Ni-NTA slurry per liter of initial expression culture.
- The suspension was packed into a gravity flow EconoPac column (SDS-PAGE sample of flow-through fraction (FT) was taken) and washed twice with 5 bed-volumes of washing buffer. SDS-PAGE samples of both washing fractions (W1 & W2) were collected.
- HTNCre-containing fractions were eluted with 3 bed-volumes of elution buffer and sample of eluate fraction (E) for SDS-PAGE analysis was taken.
- Imidazole was removed by dialyzing elution fraction against high salt buffer twice.
- The protein solution was further concentrated by dialyzing against glycerol buffer twice. In all dialysis steps the ratio of buffer to sample was at least 50. This procedure resulted in a glycerol stock solution containing HTNCre at a usual concentration between 200 and 450 μM, i.e. 1 liter of expression culture will result in ~12 mg of protein. Sample of glycerol stock (GS) for SDS-PAGE analysis was collected. HTNCre stock solution can be stored at minus 20°C.
Figure 1: SDS-PAGE analysis of the samples collected during the purification process of Cre recombinase. Induction of Cre expression is indicated by dominant band in lysate fraction. Although a part of the protein is insoluble the Cre protein can be further enriched as seen in eluate and glycerol stock fractions. L: Lysate, I: Insoluble, S: Supernatant, FT: Flow-through, W: Washing, E: Eluate, GS: Gylcerol Stock. Please click here to see a larger version of figure 1.
Protein transduction into murine embryonic stem (ES) cells:
- ES cells carrying a conditional β-Galactosidase reporter5 construct were seeded as single cells using TrypLE™ Express for dissociation of adherent cells. After 4 to 6 hours the cells had re-attached and the medium was removed.
- Subsequently ES cells were incubated with HTNCre-containing medium for 16 hours.
- An appropriate amount of HTNCre protein (corresponding to 10 μM) out of the glycerol stock was diluted into ES medium and subsequently sterile filtrated (0.22 μm).
- After protein transduction medium was changed back to normal growth medium.
- After two days cells were washed with PBS and fixed with 4% Paraformaldehyde (PFA) for 10 minutes.
- Two additional washing steps with PBS were executed before X-Gal staining was performed.
- Fixed cells were covered with a layer of X-Gal staining solution6 and incubated over night at 37°C.
Representative Results:
Next day X-Gal staining solution was aspired and the cells were covered with a layer of PBS for microscopy analysis. 80 to 100% of recombined cells could be observed within the murine ES cells judged by β-Galactosidase activity.
