Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.
The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.
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.
Purification of cell-permeable protein:
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:
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.
During the purification process of the Cre fusion protein it is important not to omit the addition of ice cold TBS buffer prior to centrifugation. Otherwise Cre recombinase tends to precipitate within the glycerol buffer.
If the eluate fraction appears to become turbid due to the high concentration of fusion protein additional elution buffer should be added until the solution has cleared again.
The application of 10 μM of Cre fusion protein typically results in a recombination efficiency of 80 to 100%. Fetal Calf Serum (FCS) being a major component of ES cell medium strongly inhibits protein transduction. Therefore high concentration of Cre recombinase had to be used. When working in serum-free conditions less protein (0.5 – 2 μM) can be used to achieve similar recombination efficiencies.
With the pSESAME vector system at hand one can apply the technique of protein transduction to other proteins including transcription factors such as Oct4 and Sox27 and Scl/Tal18.
We thank Oliver Brüstle and all members of the Stem Cell Engineering Group, University of Bonn, for support and valuable discussions. We thank Sabine Schenk for preparation of the SDS-PAGE and enduring support throughout the project. Nicole Russ and Anna Magerhans provided excellent technical support. Furthermore, we would like to thank Andreas Bär and Sheila Mertens for the production of the movie. This work was supported by grants from the Volkswagen Foundation (Az I/77864) and the German Ministry of Education and Research (BMBF, 01 GN 0813).
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
TUNER (DE3) pLacI | Novagen | 70625 | ||
Glycerol | Carl Roth | 3783.2 | ||
Na2HPO4 | Roth | T876.1 | ||
Trizma Base | Sigma-Aldrich | T1503 | ||
HCl | Roth | 4625.1 | ||
Imidazol | Roth | X998.4 | ||
NaCl | Roth | 9265.2 | ||
Yeast Extract | Roth | 2363.4 | ||
Trypton/Pepton | Roth | 8952.4 | ||
K2HPO4 | Roth | P749.2 | ||
KH2PO4 | Roth | 3904.1 | ||
Ampicillin | Sigma | A9518 | ||
Carbenicillin | Sigma | 6344.2 | ||
HEPES | Sigma | H3375 | ||
Lysozyme | Sigma | 62971 | ||
Benzonase | Novagen | |||
L-Tartaric acid, disodium salt | Sigma | |||
50% Ni-NTA slurry | Invitrogen | R901-15 | ||
EconoPac columns | Biorad | 732-1010 | ||
Sterile filter 0,22μm | Whatman | |||
Paraformaldehyde (PFA) | Sigma | |||
LB medium | Yeast extract, Trypton/Pepton, NaCl | |||
TB medium | Yeast extract, Trypton/Pepton, Glycerol, K2HPO4, KH2PO4 | |||
Lysis Buffer | 50 mM Na2HPO4, 5 mM Tris, pH 7.8 | |||
Tartaric Salt Buffer (TSB) | PTB containing 2 M L-Tartaric acid, disodium salt, and 20 mM Imidazol | |||
Washing Buffer | PTB, 500 mM NaCl, 15 mM Imidazol | |||
Elution Buffer | PTB, 500 mM NaCl, 250 mM Imidazol | |||
High Salt Buffer | 600 mM NaCl, 20 mM HEPES, pH 7.4 | |||
Gylcerol Buffer | 50% glycerol, 500 mM NaCl, 20 mM HEPES, pH 7.4 | |||
TrypLE™ Express | Invitrogen | |||
ESGRO (LIF) | Millipore | |||
NEAA | Gibco | 11140035 | ||
L-Glutamin | Gibco | 25030024 | ||
β-Mercaptoethanol | Gibco | 31350010 | ||
DMEM | Gibco | 11960044 | ||
PBS | Gibco | |||
Fetal Calf Serum (FCS) | PAA | |||
X-Gal staining solution: | 4 mM K3(FeIII(CN)6), 4 mM K4(FeII(CN)6), 2mM MgCl2 0.4 mg/mL X-Gal solved in PBS |
|||
K3(FeIII(CN)6) | Sigma | P-3367 | ||
K4 (FeII(CN)6) | Sigma | P-9387 | ||
MgCl2 | Sigma | M8266 | ||
X-Gal | Sigma | B4252 |