Name: direct protein delivery to mammalian cells using cell-permeable cys2-his2 zinc-finger domains
Uploaded: 2015-03-25T15:00:00.000+00:00
Duration: 11 min 24 s
Description: Watch this Scientific Journal Video about Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains at JoVE.com

This work was supported by the National Institutes of Health (DP1CA174426 to Carlos F. Barbas) and ShanghaiTech University, Shanghai, China (to J.L). Molecular graphics were generated using PyMol.

1. Cloning
<ol>
	<li>Obtain alanine-substituted two-finger ZiF domains that have been sub-cloned into the pET-28 expression vector system and are available upon request (pET-2F-ZiF).34</li>
	<li>PCR amplify EmGFP from the plasmid Emerald-pBAD with the primers 5&#8217; XmaI-EmGFP (5&#8217;-GGAAATTGCCCGGGATGGTGAGCAAGGGCGAGGAGCTGTTCAC-3&#8217;; XmaI site in bold) and 3&#8217; SacI-EmGFP (5&#8217;-CGGATCTGAGCTCTTACTTGTACAGCTCGTCCATGCCGAG-3&#8217;; SacI site in bold).
	<ol>
		<li>Use 5 ng of template DNA, 10 &#956;l of 10x polymerase buffer, 1 Units (U) of Taq DNA polymerase, 0.2 mM each dNTP and 0.2 &#956;M of each primer in a 100 &#956;l solution with the remaining volume made up of distilled/deionized water. Use the cycling conditions: 95 &#176;C for 5 min; 30 cycles of 95 &#176;C for 30 sec, 55 &#176;C for 30 sec and 72 &#176;C for 1 min; 72 &#176;C for 10 min. Purify the PCR product by gel extraction and determine DNA concentration using a spectrophotometer measuring Abs260 x 50 &#956;g/ml.</li>
	</ol>
	</li>
	<li>Digest pET-2F-ZiF and the insert encoding EmGFP with the restriction enzymes XmaI and SacI in recommended buffer for 3 hr at 37 &#176;C using 10 U of enzyme per 1 &#956;g of DNA. Visualize DNA by agarose gel electrophoresis using a fluorescent intercalating dye, such as ethidium bromide.</li>
	<li>Purify the digested DNA by gel extraction kit and determine DNA concentration by a spectrophotometer measuring Abs260 x 50 &#956;g/ml.</li>
	<li>Ligate the purified EmGFP-encoding DNA into 50-100 ng of pET-2F-ZiF using 1 U of T4 DNA Ligase for at least 1 h at RT. For best results, perform the ligation reaction using a 6:1 insert-to-vector molar ratio.</li>
	<li>Thaw 50 &#956;l of any chemically competent XL-1 Blue Escherichia coli cells on ice and mix gently with 10-20 ng of ligated pET-2F-ZiF-EmGFP.</li>
	<li>Keep on ice for 30 min. Heat shock the mixture at 42 &#176;C for 90 sec and recover the cells in 2 ml of Super Optimal broth with Catabolite repression (SOC) for 1 hr at 37 &#176;C with shaking.</li>
	<li>Spread 100 &#956;l of recovery culture on a lysogeny broth (LB) agar plate with 50 &#956;g/ml kanamycin and incubate O/N at 37 &#176;C.</li>
	<li>The following day, inoculate 6 ml of Super Broth (SB) or LB culture containing 50 &#956;g/ml kanamycin with one colony from the LB agar plate and culture O/N at 37 &#176;C. 
	Note: Colony PCR using the primers 5&#8217; XmaI-EmGFP and 3&#8217; SacI-EmGFP could be used to identify positive clones prior to miniprep.</li>
	<li>Purify pET-2F-ZiF-EmGFP by miniprep and confirm plasmid by DNA sequencing using T7 promoter (5&#8217;-TAATACGACTCACTATAGGG-3&#8217;).</li>
</ol>
2. Expression and Purification
<ol>
	<li>Thaw 50 &#956;l of chemically competent BL21 E. coli cells on ice and mix gently with 100 ng of pET-2F-ZiF-EmGFP plasmid. Transform as described in steps 1.7-1.8.</li>
	<li>The following day, inoculate 10 ml of LB medium containing 50 &#956;g/ml kanamycin with a single colony and grow O/N at 37 &#176;C with shaking.</li>
	<li>The following day, dilute the 10 ml of O/N culture into 1 L of LB medium supplemented with 50 &#956;g/ml kanamycin, 0.2% glucose and 100 &#956;M ZnCl2. Grow the culture at 37 &#176;C with shaking to an optical density at 600 nm (OD600) of 0.8 and induce protein expression with 2 mM isopropyl-&#946;-D-1-thiogalactopyranoside (IPTG). After 6 hr of growth at 37 &#176;C, harvest cells by centrifugation at 5,000 x g for 10 min at 4 &#176;C. 
	Note: Induction conditions are highly variable and depend on the stability of the proteins being expressed. Monitor the OD600 every 30 min until an OD600 of 0.8 is reached.</li>
	<li>Resuspend the cell pellet in 5 ml of lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 100 &#956;M ZnCl2, 1 mM dithiothreitol (DTT), 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 mM imidazole, pH 8.0). Lyse the cells on ice by sonication with the following setting: 50% power output, 4 min process time with 5 sec on/10 sec off intervals. Avoid overheating the solution.</li>
	<li>Centrifuge the cell lysate at 25,000 x g for 30 min at 4 &#176;C and transfer the supernatant into a fresh collection tube. For best results, perform all the following steps at 4 &#176;C.</li>
	<li>Run the supernatant through a column pre-packed with 1 ml of equilibrated Ni-NTA slurry. Wash the resin with 20 ml of wash buffer (50 mM Tris-HCl, 500 mM NaCl, 100 &#956;M ZnCl2, 1 mM DTT, 1 mM MgCl2 and 30 mM imidazole, pH 8.0).</li>
	<li>Elute the protein with 5 ml of elution buffer (50 mM Tris-HCl, 500 mM NaCl, 100 &#956;M ZnCl2, 1 mM DTT, 1 mM MgCl2, and 300 mM imidazole, pH 8.0).</li>
	<li>Buffer exchange the eluted protein with storage buffer (50 mM Tris-HCl, 500 mM NaCl, 100 &#956;M ZnCl2, 1 mM DTT, 1 mM MgCl2 and 10% glycerol, pH 8.0) and concentrate the protein to at least 40 &#956;M using a spin concentrator by centrifugation according to the manufacturer&#8217;s instructions.</li>
	<li>Determine protein concentration by BCA or Bradford assay. Mix 2 &#956;l of purified proteins with 2 &#956;l 2&#215; SDS-PAGE loading dye, boil at 95 &#176;C for 10 min and then resolve on 4%-20% Tris-Glycine SDS-PAGE to assess protein purity (Figure 2).</li>
</ol>
3. Protein Storage
<ol>
	<li>Aliquot the concentrated protein, flash freeze in liquid nitrogen and store at -80 &#176;C. For EmGFP fusion proteins, cover the tube with aluminum foil in order to protect the protein from light.</li>
	<li>Avoid repeated freezing and thawing of the protein solution. Note: ZiF-fused proteins are stable under these conditions for at least 1 month. Inappropriate or &#62;4 month storage may lead to protein precipitation or photobleaching of EmGFP.</li>
</ol>
4. Protein Transduction
<ol>
	<li>Maintain HeLa cells in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) and 1% antibiotic-antimycotic at 37 &#176;C in a fully humidified atmosphere with 5% CO2. 
	Note: Cells passaged more than 30 times are not recommended for protein transduction.</li>
	<li>Pre-coat a 24-well plate with 500 &#956;l of 50 &#956;g/ml of poly-lysine for 30 to 60 min at 25 &#176;C. Seed cells onto a 24-well plate at a density of 2 &#215; 105 cells per well. At 24 hr after seeding, or once cells are between 80%-90% confluent, remove media from each well and wash with 500 &#956;l of pre-warmed serum-free DMEM (SFM).</li>
	<li>To each well, add 250 &#956;l of SFM containing 2 &#956;M of ZiF-EmGFP protein and 100 &#956;M ZnCl2. Incubate cells at 37 &#176;C for 1 hr. Note: ZiF domains enter cells primarily through macropinocytosis,34 which is an energy-dependent mechanism. Therefore, cells must be incubated at 37 &#176;C for efficient protein internalization.</li>
	<li>Remove media from cells and wash three times with 500 &#956;l of calcium- and magnesium-free Dulbecco&#8217;s phosphate-buffered saline (DPBS) supplemented with 0.5 mg/ml of heparin. Note: Heparin is necessary to remove surface-bound protein that might complicate downstream analyses.</li>
	<li>(Optional) Repeat treatments up to three times for enhanced delivery.</li>
	<li>Isolate treated cells immediately after heparin wash by digestion with 0.05% trypsin-EDTA at 37 &#176;C for 2 min. Re-suspend detached cells in 0.5 ml of DPBS supplemented with 1% FBS.</li>
	<li>Measure the fluorescence intensity of each sample by flow cytometry35 using the fluorescein isothiocyanate (FITC) channel (Figure 3). Adjust the forward scatter (FSC) and side scatter (SSC) to place the population of interest on scale, ensuring that populations with different properties are resolved from each other.</li>
	<li>Collect 10,000 live events for each sample and analyze the data using flow cytometry data analysis software.36 Normalize fluorescence from HeLa cells treated with ZiF-EmGFP to those cells treated only with SFM.</li>
</ol>

<ol>
	<li>Berg, A., Dowdy, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+transduction+domain+delivery+of+therapeutic+macromolecules.">Protein transduction domain delivery of therapeutic macromolecules.</a> Curr. Opin. Biotechnol. 22, 888-893 (2011).</li><li>Lindsay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-mediated+cell+delivery:+application+in+protein+target+validation.">Peptide-mediated cell delivery: application in protein target validation.</a> Curr. Opin. Pharmacol. 2, 587-594 (2002).</li><li>Luo, D., Saltzman, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+DNA+delivery+systems.">Synthetic DNA delivery systems.</a> Nat. Biotechnol. 18, 33-37 (2000).</li><li>Guo, X., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+nonviral+vectors+for+gene+delivery.">Recent advances in nonviral vectors for gene delivery.</a> Acc. Chem. Res. 45, 971-979 (2012).</li><li>Thomas, C. E., Ehrhardt, A., Kay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+problems+with+the+use+of+viral+vectors+for+gene+therapy.">Progress and problems with the use of viral vectors for gene therapy.</a> Nat. Rev. Genet. 4, 346-358 (2003).</li><li>Frankel, A. D., Pabo, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+uptake+of+the+tat+protein+from+human+immunodeficiency+virus.">Cellular uptake of the tat protein from human immunodeficiency virus.</a> Cell. 55, 1189-1193 (1988).</li><li>Elliott, G., O'Hare, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+trafficking+and+protein+delivery+by+a+herpesvirus+structural+protein.">Intercellular trafficking and protein delivery by a herpesvirus structural protein.</a> Cell. 88, 223-233 (1997).</li><li>Derossi, D., Joliot, A. H., Chassaing, G., Prochiantz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+helix+of+the+Antennapedia+homeodomain+translocates+through+biological+membranes.">The third helix of the Antennapedia homeodomain translocates through biological membranes.</a> J. Biol. Chem. 269, 10444-10450 (1994).</li><li>Smith, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+cationic+cell-permeable+miniature+proteins+via+alpha-helical+arginine+display.">Minimally cationic cell-permeable miniature proteins via alpha-helical arginine display.</a> J. Am. Chem. Soc. 130, 2948-2949 (2008).</li><li>Daniels, D. S., Schepartz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsically+cell-permeable+miniature+proteins+based+on+a+minimal+cationic+PPII+motif.">Intrinsically cell-permeable miniature proteins based on a minimal cationic PPII motif.</a> J. Am. Chem. Soc. 129, 14578-14579 (2007).</li><li>Karagiannis, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+a+biomimetic+cell+penetrating+peptide+library.">Rational design of a biomimetic cell penetrating peptide library.</a> ACS Nan. 7, 8616-8626 (2013).</li><li>Gao, S., Simon, M. J., Hue, C. D., Morrison, B., 3r, S., Banta, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+unusual+cell+penetrating+peptide+identified+using+a+plasmid+display-based+functional+selection+platform.">An unusual cell penetrating peptide identified using a plasmid display-based functional selection platform.</a> ACS Chem. Biol. 6, 484-491 (2011).</li><li>Fuchs, S. M., Raines, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+grafting+to+endow+cell+permeability.">Arginine grafting to endow cell permeability.</a> ACS Chem. Biol. 2, 167-170 (2007).</li><li>Cronican, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+delivery+of+functional+proteins+into+mammalian+cells+in+vitro+and+in+vivo+using+a+supercharged+protein.">Potent delivery of functional proteins into mammalian cells in vitro and in vivo using a supercharged protein.</a> ACS Chem. Biol. 5, 747-752 (2010).</li><li>Panyam, J., Labhasetwar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+nanoparticles+for+drug+and+gene+delivery+to+cells+and+tissue.">Biodegradable nanoparticles for drug and gene delivery to cells and tissue.</a> Adv. Drug Deliv. Rev. 55, 329-347 (2003).</li><li>Zelphati, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+delivery+of+proteins+with+a+new+lipid-mediated+delivery+system.">Intracellular delivery of proteins with a new lipid-mediated delivery system.</a> J. Biol. Chem. 276, 35103-35110 (2001).</li><li>Kaczmarczyk, S. J., Sitaraman, K., Young, H. A., Hughes, S. H., Chatterjee, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+delivery+using+engineered+virus-like+particles.">Protein delivery using engineered virus-like particles.</a> Proc. Natl. Acad. Sci. U. S. A. 108, 16998-17003 (2011).</li><li>Voelkel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+transduction+from+retroviral+Gag+precursors.">Protein transduction from retroviral Gag precursors.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 7805-7810 (2010).</li><li>Sinha, V. R., Trehan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+microspheres+for+protein+delivery.">Biodegradable microspheres for protein delivery.</a> J. Control Release. 90, 261-280 (2003).</li><li>Liu, J., Gaj, T., Patterson, J. T., Sirk, S. J., Barbas, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-penetrating+peptide-mediated+delivery+of+TALEN+proteins+via+bioconjugation+for+genome+engineering.">Cell-penetrating peptide-mediated delivery of TALEN proteins via bioconjugation for genome engineering.</a> PLoS One. 9, e85755 (2014).</li><li>Ramakrishna, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+disruption+by+cell-penetrating+peptide-mediated+delivery+of+Cas9+protein+and+guide+RNA.">Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA.</a> Genome Res. 24, 1020-1027 (2014).</li><li>Fuchs, S. M., Raines, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyarginine+as+a+multifunctional+fusion+tag.">Polyarginine as a multifunctional fusion tag.</a> Protein Sci. 14, 1538-1544 (2005).</li><li>Mai, J. C., Shen, H., Watkins, S. C., Cheng, T., Robbins, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+of+protein+transduction+is+cell+type-dependent+and+is+enhanced+by+dextran+sulfate.">Efficiency of protein transduction is cell type-dependent and is enhanced by dextran sulfate.</a> J. Biol. Chem. 277, 30208-30218 (2002).</li><li>Al-Taei, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+traffic+and+fate+of+protein+transduction+domains+HIV-1+TAT+peptide+and+octaarginine.+Implications+for+their+utilization+as+drug+delivery+vectors.">Intracellular traffic and fate of protein transduction domains HIV-1 TAT peptide and octaarginine. Implications for their utilization as drug delivery vectors.</a> Bioconjug. Chem. 17, 90-100 (2006).</li><li>Jones, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+cell-penetrating+peptide-mediated+peptide+delivery.">Characterisation of cell-penetrating peptide-mediated peptide delivery.</a> Br. J. Pharmacol. 145, 1093-1102 (2005).</li><li>Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S., Gregory, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+with+engineered+zinc+finger+nucleases.">Genome editing with engineered zinc finger nucleases.</a> Nat. Rev. Genet. 11, 636-646 (2010).</li><li>Carroll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+with+zinc-finger+nucleases.">Genome engineering with zinc-finger nucleases.</a> Genetics. 188, 773-782 (2011).</li><li>Guo, J., Gaj, T., Barbas, C. F., 3rd, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+an+enhanced+and+highly+efficient+FokI+cleavage+domain+for+zinc+finger+nucleases.">Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases.</a> J. Mol. Biol. 400, 96-107 (2010).</li><li>Gaj, T., Guo, J., Kato, Y., Sirk, S. J., Barbas, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+knockout+by+direct+delivery+of+zinc-finger+nuclease+proteins.">Targeted gene knockout by direct delivery of zinc-finger nuclease proteins.</a> Nat. Methods. 9, 805-807 (2012).</li><li>Gersbach, C. A., Gaj, T., Barbas, C. F., 3rd, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+zinc+finger+proteins:+the+advent+of+targeted+gene+regulation+and+genome+modification+technologies.">Synthetic zinc finger proteins: the advent of targeted gene regulation and genome modification technologies.</a> Acc. Chem. Res. 47, 2309-2318 (2014).</li><li>Gaj, T., Gersbach, C. A., Barbas, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZFN,+TALEN,+and+CRISPR/Cas-based+methods+for+genome+engineering.">ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.</a> Trends Biotechnol. 31, 397-405 (2013).</li><li>Perez-Pinera, P., Ousterout, D. G., Gersbach, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+targeted+genome+editing.">Advances in targeted genome editing.</a> Curr. Opin. Chem. Biol. 16, 268-277 (2012).</li><li>Cronican, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+class+of+human+proteins+that+deliver+functional+proteins+into+mammalian+cells+in+vitro+and+in+vivo.">A class of human proteins that deliver functional proteins into mammalian cells in vitro and in vivo.</a> Chem. Biol. 18, 833-838 (2011).</li><li>Gaj, T., Liu, J., Anderson, K. E., Sirk, S. J., Barbas, C. F., 3rd, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+delivery+using+Cys2-His2+zinc-finger+domains.">Protein delivery using Cys2-His2 zinc-finger domains.</a> ACS Chem. Biol. 9, 1662-1667 (2014).</li><li>Radcliff, G., Jaroszeski, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basics+of+flow+cytometry.">Basics of flow cytometry.</a> Methods Mol. Biol. 91, 1-24 (1998).</li><li>Mercer, A. C., Gaj, T., Sirk, S. J., Lamb, B. M., Barbas, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+endogenous+human+gene+expression+by+ligand-inducible+TALE+transcription+factors.">Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.</a> ACS Synth. Biol. 3, 723-730 (2014).</li><li>Segal, D. J., Crotty, J. W., Bhakta, M. S., Barbas, C. F., Horton, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Aart,+a+designed+six-finger+zinc+finger+peptide,+bound+to+DNA.">Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA.</a> J. Mol. Biol. 363, 405-421 (2006).</li></ol>

The authors have nothing to disclose.

Here, a step-by-step protocol for protein delivery using cell-permeable zinc-finger (ZiF) domains is presented. The ZiF domain does not reduce the activity of fused enzymatic cargo34; allows for the production and purification of proteins in yields nearly identical to those observed with unmodified protein; and can transport proteins and enzymes into a wide range of cell types with efficiencies that exceed traditional cell-penetrating peptide or protein transduction domain systems. Together, these findings indicate the broad potential of ZiF domains for mediating direct protein delivery into cells for a wide range of applications.
Maximum protein delivery was previously achieved using only a two-finger ZiF domain, despite the fact that extended arrays of three- and four-finger ZiF domains carry greater positive charge. These findings indicate that ZiF-mediated cell entry might be influenced by factors other than charge, including protein stability or conformational rigidity. ZiF domain-mediated protein delivery was also found to be energy-dependent and thus requires that all cells treated with protein be incubated at 37 &#176;C. Through the usage of small molecule inhibitors of various endocytic pathways, macropinocytosis, and to a lesser extent caveolin-dependent endocytosis, were determined to be the major pathways for ZiF-mediated cell entry.34 Notably, unlike other protein transduction systems, ZiF domains are capable of efficiently escaping endosomes to mediate high levels of cytosolic delivery of the fused macromolecular cargo, underscoring the potential of these domains for achieving robust protein delivery.
In our experience, the seeding density of cells is a critical step for achieving high levels of protein transduction. We recommend treating cells once they reach 80%-90% confluency and previously observed that cells seeded at &#62;95% confluency show sub-optimal transduction capacity, while cells seeded at low densities (&#60;50%) are susceptible to protein-induced toxicity. Importantly, for cell types with high detachment tendencies, cell culture plates pre-coated with poly-lysine are recommended. Poly-lysine facilitates cell attachment through electrostatic interactions with negatively charged cell-surface components. Although the &#945;-helical DNA-binding residues of each ZiF domain have been removed to eliminate any potential for DNA recognition, the cysteine and histidine residues that coordinate with the Zn2+ ion to stabilize the &#914;&#914;&#945; ZiF domain fold remain intact. Thus, we recommend that any storage buffer be supplemented with at least 100 &#956;M of ZnCl2 to maintain protein integrity.
Although the ZiF domain was previously shown to deliver proteins and enzymes into a variety of cell types, the efficiency of ZiF delivery might also be dependent on the both macromolecular cargo and protein concentration. For instance, cells treated with two-finger ZiF-luciferase fusion protein were observed to display maximum luminescence when treated with 0.5 &#956;M protein, with decreased activity at higher concentrations, while cells treated with two-finger EmGFP exhibited a dose-dependent increase in fluorescence intensity up to 8.0 &#956;M protein, and upon consecutive protein treatments. We therefore recommend evaluating the cell penetrating ability of each unique ZiF domain fusion across a range of concentrations.
Finally, although not yet demonstrated, we anticipate that ZiF domain delivery is a highly flexible platform, capable of delivering a diverse array of macromolecules into cells. For example, it may be possible for both DNA and RNA to be chemically functionalized onto the surface of the ZiF domain via a hydrolyzable linker, or transiently transfected into cells by encapsulation of ZiF domains. Additionally, the efficiency of ZiF protein delivery could be further enhanced by rational design efforts focused on surface charge optimization.

Highly efficient and versatile protein delivery strategies are critical for many basic research and therapeutic applications. The direct delivery of purified proteins into cells represents one of the safest and easiest methods for achieving this.1,2 Unlike strategies that rely on gene expression from nucleic acids,3-5 protein delivery poses no risk of insertional mutagenesis, is independent of the cellular transcription/translation machinery and allows for an immediate effect. However, the lack of simple and generalizable methods for endowing cell-penetrating activity onto proteins routinely confounds their direct entry into cells. Current methods for facilitating intracellular protein delivery are based on the use of naturally occurring6-8 or designed cell-penetrating peptides,9-12 supercharged transduction domains,13,14 nanoparticles15 and liposomes,16 virus-like particles17,18 and polymeric microsphere materials.19 Unfortunately, many of these approaches are hampered by low cellular uptake rates,20,21 poor stability,22 inadvertent cell-type specificity,23 low endosomal escape properties24 and toxicity.25 In addition, many protein transduction technologies reduce the bioactivity of the delivered proteins.14
Our laboratory previously demonstrated that zinc-finger nuclease (ZFN) proteins &#8212; chimeric restriction endonucleases consisting of a programmable Cys2-His2 zinc-finger DNA-binding protein and the cleavage domain of the FokI restriction endonuclease26-28&#160;&#8212; are inherently cell-permeable.29 This surprising cell-penetrating activity was shown to be an intrinsic property of the custom-designed zinc-finger domain, a DNA-binding platform that has emerged as a powerful tool for targeted genome engineering,30-32 and considered to be the result of the constellation of six positively charged residues on the protein surface. Indeed, several DNA-binding proteins, including c-Jun and N-DEK have been shown to possess an innate capacity to cross cell membranes.33 More recently, our laboratory expanded on these results and demonstrated that the cell-penetrating activity of zinc-finger (ZiF) domains could be leveraged for intracellular protein delivery. Genetic fusion of either one- or two-finger ZiF domains to specific protein cargo led to uptake efficiencies that exceeded many conventional cell-penetrating peptide delivery systems.34 Most notably, ZiF-mediated delivery did not compromise the activity of fused enzymatic cargo and facilitated high levels of cytosolic delivery. Collectively, these findings demonstrate the potential of the ZiF domain for facilitating the efficient and facile delivery of proteins, and potentially more diverse types of macromolecules, into cells.
Here, a detailed step-by-step protocol on how to implement ZiF technology for protein delivery in mammalian cells is presented. We previously constructed a suite of one-, two-, three-, four-, five- and six-finger ZiF domains that lack the ability to bind DNA, due to substitution of each of the &#945;-helical DNA-binding residues, but are capable of delivering proteins into cells34 (Figure 1). The production and transduction of Emerald GFP (EmGFP) into HeLa cells using a two-finger ZiF domain is described. This protocol is extensible to almost any protein capable of soluble expression in Escherichia coli and nearly any mammalian cell type. Expected results are provided and strategies for maximizing the performance of this system are also discussed.

<table><tbody><tr><td>XmaI</td><td>New England Biolabs</td><td>R0180L</td><td></td></tr><tr><td>SacI</td><td>New England Biolabs</td><td>R0156L</td><td></td></tr><tr><td>Expand High Fidelity PCR system</td><td>Roche</td><td>11759078001</td><td></td></tr><tr><td>dNTPs</td><td>New England Biolabs</td><td>N0446S</td><td></td></tr><tr><td>4%-20% Tris-Glycine Mini protein gels, 1.5 mm, 10 wells</td><td>Life Technologies</td><td>EC6028BOX</td><td></td></tr><tr><td>2x Laemmli Sample Buffer</td><td>BioRad</td><td>161-0737</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>Life Technologies</td><td>15224-017</td><td></td></tr><tr><td>BL21 (DE3) Competent &lt;em&gt;E. coli&lt;/em&gt;</td><td>New England Biolabs</td><td>C2527I</td><td></td></tr><tr><td>IPTG</td><td>Thermo Scientific</td><td>R0391</td><td></td></tr><tr><td>Zinc Chloride</td><td>Sigma-Aldrich</td><td>208086-5G</td><td></td></tr><tr><td>Kanamycin Sulfate</td><td>Fisher Scientific</td><td>BP906-5</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270-100G</td><td></td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP152-25</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>S9888-25G</td><td></td></tr><tr><td>DTT</td><td>Fisher Scientific</td><td>PR-V3151&amp;nbsp;</td><td></td></tr><tr><td>PMSF</td><td>Thermo Scientific</td><td>36978</td><td></td></tr><tr><td>Ni-NTA Agarose Resin</td><td>QIAGEN</td><td>30210</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516-500ML</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I5513-25G</td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Units</td><td>EMO Millipore</td><td>UFC900324</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11966-025</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>10437-028</td><td></td></tr><tr><td>Antibiotic-Antimycotic&amp;nbsp;</td><td>Life Technologies</td><td>15240-062</td><td></td></tr><tr><td>24-Well Flat Bottom Plate</td><td>Sigma-Aldrich</td><td>CLS3527-100EA</td><td></td></tr><tr><td>Poly-Lysine</td><td>Sigma-Aldrich</td><td>P7280</td><td></td></tr><tr><td>DPBS, No Calcium, No Magnesium</td><td>Life Technologies</td><td>21600010</td><td></td></tr><tr><td>Heparan Sulfate</td><td>Sigma-Aldrich</td><td>H4777</td><td></td></tr><tr><td>Trypsin</td><td>Life Technologies</td><td>25300054</td><td></td></tr><tr><td>HeLa cells</td><td>ATCC</td><td>CCL-2</td><td></td></tr><tr><td>Nano Drop ND-1000 spectrophotometer&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>QIAGEN</td><td>28104</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>QIAGEN</td><td>28704</td><td></td></tr></tbody></table>

direct protein delivery to mammalian cells using cell-permeable cys2-his2 zinc-finger domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Two-finger ZiF-EmGFP fusion proteins can be expressed in E. coli with &#62;95% homogeneity and high yields (&#62;25 mg/ml) (Figure 2). In general, one- and two-finger ZiF fusion proteins can be produced in quantities nearly identical to those of wild-type unmodified protein. However, in some contexts, five- and six-finger ZiF fusion proteins are unable to be produced in yields high enough for downstream applications.
Direct application of two-finger ZiF-EmGFP protein onto HeLa cells for 90 min at 37 &#176;C leads to a dose-dependent increase in EmGFP fluorescence (Figure 3A). Critically, no fluorescence is observed in the absence of the ZiF domain. We previously observed that nearly 100% of cells are fluorescent after treatment with only 2 &#956;M of two-finger ZiF-EmGFP protein, and that HeLa cells treated with ZiF fusion protein are positive for EmGFP fluorescence at protein concentrations as low as 0.25 &#956;M (Figure 3B).
<img alt="Figure 1" src="/files/ftp_upload/52814/52814fig1.jpg" /> 
Figure 1. Structure and sequence of zinc-finger protein. (Top) Crystal structure of a single zinc-finger (ZiF) domain. The side chains of the conserved Cys and His residues coordinated with the Zn2+ ion are shown as sticks (PDB ID: 2I13).37 (Bottom) Sequence of the ZiF domain. Arrows and cylinders indicate &#914;-sheet and &#945;-helix secondary structures, respectively. The &#945;-helical DNA-binding residues that have been substituted with alanine are highlighted pink. Positively charged residues predicted to mediate cell internalization are highlighted light blue. <a href="https://www.jove.com/files/ftp_upload/52814/52814fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52814/52814fig2.jpg" /> 
Figure 2. SDS-PAGE of purified one-, two-, three- and four-finger ZiF-EmGFP fusion proteins. ZiF-EmGFP fusion proteins were expressed in E. coli and purified by Ni-NTA agarose resin. Eluted protein was analyzed for purity by SDS-PAGE using a 4%-20% Tris-Glycine gel. No significant degradation or truncations of ZiF-EmGFP fusion proteins was observed.
<img alt="Figure 3" src="/files/ftp_upload/52814/52814fig3.jpg" /> 
Figure 3. ZiF-mediated protein delivery into HeLa cells. (A) Fluorescence intensity of HeLa cells treated with increasing amounts of two-finger ZiF-EmGFP protein. HeLa cells treated with EmGFP protein alone overlap entirely with untreated cells. (B) Normalized fluorescence intensity of HeLa cells treated consecutively with 2 &#956;M of two-finger ZiF-EmGFP protein. Fluorescence intensity was determined by flow cytometry.

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Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

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Biology

9.9K Views.  The Scripps Research Institute. Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Video: Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Genetics

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Biochemistry

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Bioengineering

Engineering Intracellular Protein Sensors in Mammalian Cells

Proteins can function as biomarkers of pathological conditions, such as neurodegenerative diseases, infections or metabolic syndromes. Engineering cells to sense and respond to these biomarkers may help the understanding of molecular mechanisms underlying pathologies, as well as to develop new cell-based therapies. While several systems that detect extracellular proteins have been developed, a modular framework that can be easily re-engineered to sense different intracellular proteins was missing.
Here, we describe a protocol to implement a modular genetic platform that senses intracellular proteins and activates a specific cellular response. The device operates on intracellular antibodies or small peptides to sense with high specificity the protein of interest, triggering the transcriptional activation of output genes, through a TEV protease (TEVp)-based actuation module. TEVp is a viral protease that selectively cleaves short cognate peptides and is widely used in biotechnology and synthetic biology for its high orthogonality to the cleavage site. Specifically, we engineered devices that recognize and respond to protein-biomarkers of viral infections and genetic diseases, including mutated huntingtin, NS3 serine-protease, Tat and Nef proteins to detect Huntington&#8217;s disease, hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections, respectively. Importantly, the system can be hand tailored for the desired input-output functional outcome, such as fluorescent readouts for biosensors, stimulation of antigen presentation for immune response, or initiation of apoptosis to eliminate unhealthy cells.

Here, we present a protocol for engineering genetically-encoded intracellular protein sensor-actuator(s). The device specifically detects target proteins through intracellular antibodies (intrabodies) and responds by switching on gene transcriptional output. A general framework is built to rapidly replace intrabodies, enabling rapid detection of any desired protein, without altering the general architecture.

Proteins can function as biomarkers of pathological conditions, such as neurodegenerative diseases, infections or metabolic syndromes. Engineering cells ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Engineering Cell-permeable Protein

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.

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 ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys₂-His₂ Zinc-finger Domains

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