This research was supported by Cleveland State University Faculty Development Funds.

1. Preparation of Parasite Cell Cultures, Collection of Parasite Pellets
<ol>
	<li>Prepare RPMI-1640-HEPES media by adding 10 g of RPMI-1640 powder, 66 mg of gentamycin sulphate, 2 g sodium bicarbonate, 5.9 g HEPES buffer, 50 mg hypoxanthine and 3.8 g dextrose in 1 L sterile dH2O. Mix using table magnetic stirrer until all the powder dissolves in 1 L distilled water. Perform micro filtration of media using 0.22 &#181;m filter. Store the media in sterile bottles.</li>
	<li>Wash uninfected (fresh) type A+ erythrocytes using RPMI. Prepare 5% hematocrit of erythrocytes (0.5 ml of washed fresh erythrocytes in 9.5 ml 10% RPMI + human serum).</li>
	<li>Make 15% human serum + RPMI (15 ml of human serum and 85 ml of RPMI) to initiate the cultivation of parasites. Make 10% human serum and RPMI (10 ml of human serum and 90 ml of RPMI) to continue culturing of parasites in red blood cells.</li>
	<li>Culture P. falciparum (3D7 and FCR-3 strains) in type A+ human erythrocytes at 5% hematocrit and 20% parasitemia in petri dish or a 75 cm3 culture flask. Maintain culture in vitro according to the method of Trager and Jensen candle jar26. Alternately, maintain cultures are in incubator at 37 &#176;C maintaining 9.5% CO2 and 3.4% N2 gas. Culture P. falciparum in RPMI 1640-HEPES media supplemented with 10% human serum.</li>
	<li>Synchronize P. falciparum culture by adding 65% percoll (65 ml of percoll + 35 ml of RPMI-1640 without human serum) to the culture concentrate27.</li>
	<li>Centrifuge the culture concentrate with percoll at 2500 x g for 5 min which results in 3 layers. Carefully, using a transfer pipette isolate schizonts from the middle layer transfer into a separate tube under aseptic conditions27.</li>
	<li>Wash the isolated cultures with 10% human serum + RPMI-1640 twice to remove excess percoll by centrifuging at 7500 x g for 5 min. Discard the supernatant each time under aseptic conditions27.</li>
	<li>Continue culturing the synchronized P. falciparum schizonts in 10% human serum + RPMI-164027.</li>
	<li>Collect schizonts by treating Plasmodium-infected erythrocytes with 10 mM Tris pH 8.8 and centrifuging at 15000 x g for 15 min23. Store parasite pellets at -70 &#176;C following addition of 5 &#181;l of protease inhibitor (aprotinin) to the pellets. Use parasite pellets for protein extraction, genomic DNA isolation and RNA isolation.</li>
</ol>
2. Preparing Plasmid Vector
<ol>
	<li>Isolate genomic DNA from P. falciparum schizont pellets prepared from cultures of approximately 20% parasitemia.</li>
	<li>Add 600 &#181;l of 10mM Tris-HCl of pH 7.6, 50 mM EDTA pH 8.0, 0.1% SDS and 1 mg/ml proteinase K, to pellets in a microcentrifuge tube, homogenize pellet using a 1 ml syringe attached to a 26 G needle. Alternate filling the syringe with the pellet mixture and emptying into a microcentrifuge tube 20 times. Incubate tube containing homogenate O/N in a 50 &#176;C water bath.</li>
	<li>Perform phenol &#8211; chloroform (P+C) extraction by adding 600 &#181;l of P+C (300 &#181;l of phenol and 300 &#181;l of chloroform) to homogenized pellet. Cap tube tightly and shake the mixture vigorously by inverting tube end-to-end. Centrifuge tube at 14,000 x g for 2 min and collect upper aqueous solution using a micropipette into a new microcentrifuge tube. Repeat this step twice.</li>
	<li>Add 600 &#181;l of chloroform to the aqueous layer in the new tube from step 2.1.2. Vortex the tube for 10 sec and centrifuge at 14,000 x g for 2 min. Measure the upper aqueous layer with a micropipette and transfer it to a new microcentrifuge tube.</li>
	<li>Add 0.1 vol of 5 M sodium acetate pH 5.5 and 1 vol of isopropanol (if aqueous layer is 300 &#181;l add 30 &#181;l of 5 M sodium acetate pH 5.5 + 330 &#181;l of isopropanol) to aqueous layer from step 2.1.3. Incubate the tube at RT for 15 min.</li>
	<li>Centrifuge the tube at 14,000 x g for 10 min to precipitate DNA pellet. Discard the supernatant and wash pellet with 100 &#181;l of 70% ethanol (70 &#181;l of ethanol and 30 &#181;l of dH2O) to remove excess salt from the DNA. Store DNA in 50 &#181;l of dH2O at -20 &#176;C.</li>
	<li>Design forward and reverse primers using software or manually for amplification of P. falciparum genes PF3D7_1361800, PF3D7_0925900, PF3D7_1436300 and PF3D7_0114100 previously identified in proteome studies16,18 with appropriate restriction sites to permit cloning of the gene sequences into the multiple cloning sites of expression plasmid pT7CFE1-CHis as shown in Table 2.</li>
	<li>Make a 50 &#181;l reaction mixture by adding; 5 &#181;l of isolated P. falciparum genomic DNA, 5 &#181;l of 10x buffer, 5&#181;l of MgCl2, 1.5 &#181;l of forward primer, 1.5 &#181;l of reverse primer, 0.5 &#181;l of T7 DNA polymerase and 31.5 &#181;l of dH2O and perform polymerase chain reaction (PCR) at 94 &#176;C for 8 min for denaturation, 50 &#176;C for 1 min 30 sec for extension and 72 &#176;C for 9 min for annealing and renaturation for amplifying genes shown in Table 1.</li>
	<li>Separate PCR products on 1% agarose gel (1 g of agarose and 100 ml of Tris acetate EDTA (TAE) buffer)32.</li>
	<li>Cut the PCR amplified DNA bands from the agarose gel, place the gel slices containing DNA in a DNA gel extraction spin column and freeze at -20 &#176;C for 5 min. Add 100 &#181;l of isopropanol to the frozen gel slices and centrifuge at 14,000 x g for 5 min.</li>
	<li>Measure the aqueous flow-through filtered in the collection tube from step 2.5, using a micropipette and transfer to a new tube. Add 0.1 vol of 5 M sodium acetate and centrifuge at 14,000 x g for 5 min. Discard the supernatant and add 10 &#181;l of dH2O to the DNA pellet.</li>
	<li>Digest the pT7CFE1-CHis expression vector and purified DNA fragments (from 2.6) in two separate tubes by adding 3 &#181;l of plasmid and 5 &#181;l of PCR gene products to each tube. Add to each tube, 1 &#181;l of specific restriction enzymes, 2 &#181;l of reaction buffer and 14 &#181;l of dH2O.</li>
	<li>Add 3 &#181;l of digested plasmid, 5 &#181;l of digested DNA fragments, 2 &#181;l of 10x ligase buffer, 1 &#181;l of ligase and 9 &#181;l of dH2O in an microcentrifuge tube. Incubate at 12 &#176;C O/N.</li>
	<li>Add 0.1 vol of 3 M sodium acetate at pH 5.5 and 2 volumes of ethanol (if your ligation tubes volume is 20 &#181;l add 2 &#181;l of 3 M sodium acetate + 44 &#181;l of ethanol) to the tube from step 2.14. Mix well and incubate at -20 &#176;C for 15 min.</li>
	<li>Centrifuge the tube at 14,000 x g for 15 min. Carefully remove the supernatant using a micro pipette without disturbing the pellet.</li>
	<li>Add 100 &#181;l of 70% ethanol to the pellet from step 2.10. Centrifuge the tube at 14,000 x g for 5 min. Remove and discard the supernatant. Add 10 &#181;l of nuclease-free water to the DNA pellet.</li>
</ol>
3. Protein Expression Using in Vitro Human Cell Free Expression System
<ol>
	<li>Prepare 20 &#181;l of transcription mixture by measuring 2 &#181;l of the recombinant pT7CFE1-CHis plasmid DNA, 4 &#181;l of 5x transcription buffer, 4 &#181;l of NTP mix, 2 &#181;l of T7 RNA Polymerase and 8 &#181;l of nuclease-free water for 2-step Human in Vitro Protein Expression using DNA templates. Mix components in a microcentrifuge tube and incubate for 75 min at 30 &#176;C in a water bath.</li>
	<li>Take 2 &#181;l of the transcription mixture and add it to 23 &#181;l of translation mixture containing 12.5 &#181;l of HeLa cell lysate, 2.5 &#181;l of accessory proteins, 1 &#181;l of salt solution A, 0.5 &#181;l of amino acids &#8211;Met, 0.5 &#181;l of amino acids &#8211;Leu, 1 &#181;l of RNAse inhibitor, 1.25 &#181;l of energy mix and 3.75 &#181;l of nuclease-free water.</li>
	<li>Incubate the translation mixture from step 3.2 at 30 &#176;C for 90 min. Store translated products at -20 &#176;C.</li>
	<li>Prepare 25 &#181;l of transcription and translation mixture by adding 3 &#181;l of the recombinant pT7CFE1-CHis plasmid DNA, 2 &#181;l of nuclease-free water, 5 &#181;l of reaction mix, 12.5 &#181;l of HeLa lysate and 2.5 &#181;l of accessory proteins for 1-step Human Coupled IVT Protein Expression for DNA templates.</li>
	<li>Incubate the reaction mixture from step 3.4 for 90 min to 6 hr at 30 &#176;C. Store the translation products at -20 &#176;C.</li>
</ol>
4. Purification of Expressed Recombinant Proteins
<ol>
	<li>Add 75 &#181;l of 1x purification buffer to 25 &#181;l of translation product to make 100 &#181;l of purification mixture.</li>
	<li>Add 100 &#181;l of nickel chelating resin to 100 &#181;l of purification mixture. Wash Ni-resin twice by adding 300 &#181;l of dH2O and 300 &#181;l of the binding buffer. Re-suspend resin and centrifuge at 14,000 x g for 1 min to remove excess ethanol.</li>
	<li>Add 100 &#181;l of purification mixture from step 4.2 to 100 &#181;l of nickel chelating resin and incubate the mixture for 60 min at RT.</li>
	<li>Centrifuge the mixture at 14,000 x g for 1 min and collect supernatant into a fresh tube labeled as &#8220;flow through&#8221;.</li>
	<li>Wash resin with 100 &#181;l of 1x wash buffer (20 mM imidazole, 250 mM NaH2PO4 at pH 8, 2.5 M NaCl and dH2O). Centrifuge at 14,000 x g for 1 min and collect supernatant in fresh tube labeled &#8220;wash&#8221;. Repeat step 4.5 twice.</li>
	<li>Add 100 &#181;l of 1x elution buffer (100 mM Imidazole, 250 mM Na2PO4 at pH 8.0, 2.5M NaCl and dH2O) to the resin and incubate for 15 min. Centrifuge at 14,000 x g for 1 min. Do the elution step twice. Each time collect supernatant into fresh microcentrifuge tubes and label as &#8220;eluate&#8221;.</li>
	<li>After elution, wash resin twice as described in step 4.5. Store flow through, washes and elution samples at -20 &#176;C or lower. Resin must be stored at 4 &#176;C. Use 30 &#181;l of each sample for SDS-PAGE and immunoblotting analysis.</li>
</ol>
5. Coomassie (Bradford) Protein Assay 34
<ol>
	<li>Prepare standard bovine serum albumin (BSA) solutions with the concentration 20 &#181;g/ml (mix 10 &#181;l of BSA (2 mg/ml) and 90 &#181;l of dH2O), 40 &#181;g/ml (mix 20 &#181;l of BSA (2 mg/ml) and 80 &#181;l of dH2O), 60 &#181;g/ml (mix 30 &#181;l of BSA (2 mg/ml) and 70 &#181;l of dH2O), 80 &#181;g/ml (mix 40 &#181;l of BSA (2 mg/ml) and 60 &#181;l of dH2O), 100 &#181;g/ml (mix 50 &#181;l of BSA (2 mg/ml) and 50 &#181;l of dH2O), 160 &#181;g/ml (mix 80 &#181;l of BSA (2 mg/ml) and 20 &#181;l of dH2O) and 200 &#181;g/ml (100 &#181;l of BSA (2 mg/ml).</li>
	<li>Measure 5 &#181;l of flow through, washes and eluate samples from step 4.7. Add 95 &#181;l of dH2O.</li>
	<li>Add 5 ml of Coomassie blue reagent to the mixtures from steps 5.1 and 5.2. Cover the tubes with parafilm and vortex for 10 sec then incubate for 20 min at RT.</li>
	<li>Using a spectrophotometer, measure optical density (OD) at wavelength of 650 nm for the protein tubes from step 5.3. Plot concentration vs. OD reading to determine the protein concentrations34.</li>
</ol>
6. Western Blotting
<ol>
	<li>Add 5 &#181;l of schizont extracts from P. falciparum, 2 &#181;l of Escherichia coli expressed recombinant Rhop-3 proteins31, 5 &#181;l of recombinant proteins expressed using 2-step and 1-step in vitro human expression systems and 10 &#181;l of protein products purified using affinity method to separate tubes.</li>
	<li>Add electrophoresis sample buffer containing mercaptoethanol to each tube for a final volume of 20 &#181;l to make solubilized protein samples. Boil tubes for 2 min.</li>
	<li>Load solubilized protein samples onto 10% SDS-PAGE gels to separate the proteins.</li>
	<li>Transfer separated proteins from SDS-PAGE gels to nitrocellulose paper (NCP) by electrophoresing at 35 mA current per gel in a semi-dry western blotting chamber for 2 hr.</li>
	<li>Block NCPs following transfer with 2% nonfat milk.</li>
	<li>Incubate blocked NCPs with the following polyclonal antibodies diluted 1:100 in 2% milk to perform western blotting; rabbit antibody #67621 specific for P. falciparum merozoite rhoptries, mouse antibodies20 specific for P. yoelii and P. berghei merozoite rhoptries, antisera #685 specific for the P. falciparum parasitophorous vacuole protein, SERA (serine rich antigen)22 and antibodies specific for the Maurer&#8217;s cleft protein PfMC-2TM28.</li>
	<li>Incubate NCPs also in 1:100 diluted normal mouse and rabbit serum and spent culture supernatant (SCS) from SP2 myeloma cells as negative controls.</li>
	<li>Incubate NCPs with antibodies and controls at 4 &#186;C O/N.</li>
	<li>Wash NCPs 4x with Blot buffer and incubate NCPs with species specific secondary antibodies conjugated to horseradish peroxidase (HRP) diluted 1:1000 in 2% milk.</li>
	<li>Wash NCPs 4x with Blot buffer. Incubate washed NCPs with the color development solution A+B (Solution A: add 50ml of 1x Blot buffer and 30 &#181;l of H2O2; Solution B: add 10 ml of methanol and 30 mg of 3-chloro-naphtol) for 30 min in dark. Analyze western blot results colorimetrically.</li>
</ol>

<ol>
	<li>Alexandrov, K., 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=Leishmania.+cell-free+expression+system.">Leishmania. cell-free expression system.</a> Methods. 55 (1), 58-64 (2011).</li><li>Alexandrov, K., 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=Towards+the+construction+of+expressed+proteomes+using+a+Leishmania+tarentolae.+based+cell-free+expression+system.">Towards the construction of expressed proteomes using a Leishmania tarentolae. based cell-free expression system.</a> PLOS One. 5 (12), e14388 (2010).</li><li>Baca, A. M., Hol, W. G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+codon+bias:+A+method+for+high-level+over-expression+of+Plasmodium.+and+other+AT-rich+parasite+genes+in+Escherichia+coli.">Overcoming codon bias: A method for high-level over-expression of Plasmodium. and other AT-rich parasite genes in Escherichia coli.</a> Int J Parasitol. 30 (2), 113-118 (2000).</li><li>Birkholtz, L. M., 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=Heterologous+expression+of+plasmodial+proteins+for+structural+studies+and+functional+annotation.">Heterologous expression of plasmodial proteins for structural studies and functional annotation.</a> Malar. J. 7, 197 (2008).</li><li>Doolan, D. L., Mu, Y., Unal, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+humoral+immune+responses+to+P.+falciparum.+infection+with+protein+microarrays.">Profiling humoral immune responses to P. falciparum. infection with protein microarrays.</a> Proteomics. 8 (22), 4680-4694 (2008).</li><li>Endo, Y., Sawasaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+expression+systems+for+eukaryotic+protein+production.">Cell-free expression systems for eukaryotic protein production.</a> Curr. Opin. Biotechnol. 17 (4), 373-380 (2006).</li><li>Fern&#225;ndez-Robledo, J. A., Vasta, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+proteins+from+protozoan+parasites.">Production of recombinant proteins from protozoan parasites.</a> Trends Parasitol. 26 (5), 244-254 (2010).</li><li>Gowda, D. C., Davidson, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+glycosylation+in+the+malaria+parasite.">Protein glycosylation in the malaria parasite.</a> Parasitol. Today. 15 (4), 147-152 (1999).</li><li>Goshima, N., 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=Human+protein+factory+for+converting+the+transcriptome+into+an+in+vitro-expressed+proteome.">Human protein factory for converting the transcriptome into an in vitro-expressed proteome.</a> Nat. Methods. 5, 1011-1017 (2008).</li><li>Hinnebusch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mechanism and regulation of initiator methionyl-tRNA binding to ribosomes. 5, 185-243 (2000).</li><li>Hino, M., 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=Efficiency+of+cell-free+protein+synthesis+based+on+a+crude+cell+extract+from+Escherichia+coli.,+wheat+germ,+and+rabbit+reticulocytes.">Efficiency of cell-free protein synthesis based on a crude cell extract from Escherichia coli., wheat germ, and rabbit reticulocytes.</a> Journal of Biotechnology. 133 (2), 183-189 (2008).</li><li>Le Roch, K. G., 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=Discovery+of+gene+function+by+expression+profiling+of+the+malaria+parasite+life+cycle.">Discovery of gene function by expression profiling of the malaria parasite life cycle.</a> Science. 301 (5639), 1503-1508 (2003).</li><li>Makrides, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+achieving+high-level+expression+of+genes+in+Escherichia+coli.">Strategies for achieving high-level expression of genes in Escherichia coli.</a> Microbiol. Rev. 60 (3), 512-538 (1996).</li><li>Mehlin, 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=Heterologous+expression+of+proteins+from+Plasmodium+falciparum.:+results+from+1000+genes.">Heterologous expression of proteins from Plasmodium falciparum.: results from 1000 genes.</a> Mol. Biochem. Parasitol. 148 (2), 144-160 (2006).</li><li>Mikami, 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=A+human+cell-derived+in+vitro+coupled+transcription/translation+system+optimized+for+production+of+recombinant+proteins.">A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins.</a> Protein Expr. Purif. 62 (2), 190-198 (2008).</li><li>Sam-Yellowe, T. Y., 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=Proteome+analysis+of+rhoptry:+Enriched+isolated+from+Plasmodium.+merozoites.">Proteome analysis of rhoptry: Enriched isolated from Plasmodium. merozoites.</a> Journal of Proteome Research. 3 (5), 995-1001 (2004).</li><li>Sam-Yellowe, T. Y., Fujioka, H., Aikawa, M., Hall, T., Drazbar, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum.+protein+located+in+Maurer&#8217;s+clefts+underneath+knobs+and+protein+localization+in+association+with+Rhop-3+and+SERA+in+the+intracellular+network+of+infected+erythrocytes.">Plasmodium falciparum. protein located in Maurer&#8217;s clefts underneath knobs and protein localization in association with Rhop-3 and SERA in the intracellular network of infected erythrocytes.</a> Parasitol Res. 87 (3), 173-185 (2001).</li><li>Sam-Yellowe, T. Y., 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+Plasmodium.+gene+family+encoding+Maurer&#8217;s+cleft+membrane+proteins:+Structural+properties+and+expression+profiling.">A Plasmodium. gene family encoding Maurer&#8217;s cleft membrane proteins: Structural properties and expression profiling.</a> Genome Res. 14 (6), 1052-1059 (2004).</li><li>Sam-Yellowe, T. Y., Banks, T. L., Fujioka, H., Drazba, J. A., Yadav, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+yoelii.:+Novel+rhoptry+proteins+identified+within+the+body+of+merozoite+rhoptries+in+rodent+Plasmodium+malaria.">Plasmodium yoelii.: Novel rhoptry proteins identified within the body of merozoite rhoptries in rodent Plasmodium malaria.</a> Exp Parasitol. 120 (1), 113-117 (2008).</li><li>Sam-Yellowe, T. Y., Rio, D., Fujioka, H., Aikawa, M., Yang, J. C., Yakubu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+merozoite+rhoptries,+identification+of+novel+rhoptry+associated+proteins+from+P.+yoelii.,+P.+chabaudi.,+P.+berghei.+and+conserved+interspecies+reactivity+of+organelles+and+proteins+with+P.+falciparum.+rhoptry-specific+antibodies.">Isolation of merozoite rhoptries, identification of novel rhoptry associated proteins from P. yoelii., P. chabaudi., P. berghei. and conserved interspecies reactivity of organelles and proteins with P. falciparum. rhoptry-specific antibodies.</a> Exp Parasitol. 89 (3), 271-284 (1998).</li><li>Sam-Yellowe, T. Y., Fujioka, H., Masamichi, A., Messineo, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum.+rhoptry+proteins+of+140/130/110kd+(Rhop-H)+are+located+in+an+electron+lucent+compartment+in+the+neck+of+the+rhoptries.">Plasmodium falciparum. rhoptry proteins of 140/130/110kd (Rhop-H) are located in an electron lucent compartment in the neck of the rhoptries.</a> J Euk Microbial. 42 (3), 224-231 (1995).</li><li>Sam-Yellowe, T. Y., Hishashi, F., Masamichi, A., Messineo, D. G., Leash, A. M., Hall, T., Drazba, J. A., Ndengele, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Organization+and+cross+linking+analysis+of+the+Plasmodium+falciparum+erythrocyte+binding+proteins+Rhop-H+and+SERA.">Molecular Organization and cross linking analysis of the Plasmodium falciparum erythrocyte binding proteins Rhop-H and SERA.</a> J Protozool. Res. 10 (3), 128-154 (2000).</li><li>Sam-Yellowe, T. Y., Shio, H., Perkins, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretion+of+Plasmodium+falciparum.+rhoptry+protein+into+plasma+membrane+of+host+erythrocytes.">Secretion of Plasmodium falciparum. rhoptry protein into plasma membrane of host erythrocytes.</a> J Cell Biol. 106 (5), 1507-1513 (1988).</li><li>Sundaresh, 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=Identification+of+humoral+immune+responses+in+protein+microarrays+using+DNA+microarray+data+analysis+techniques.">Identification of humoral immune responses in protein microarrays using DNA microarray data analysis techniques.</a> Bioinformatics. 22 (14), 1760-1766 (2006).</li><li>Terpe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+bacterial+expression+systems+for+heterologous+protein+production:+from+molecular+and+biochemical+fundamentals+to+commercial+systems.">Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems.</a> Appl. Microbiol. Biotechnol. 72 (2), 211-222 (2006).</li><li>Trager, W., Jensen, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+malaria+parasites+in+continuous+culture.">Human malaria parasites in continuous culture.</a> Science. 193 (4254), 673-675 (1976).</li><li>Tsarukyanova, I., Drazba, J. A., Fujioka, H., Yadav, S., Sam-Yellowe, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteins+of+the+Plasmodium+falciparum.+two+transmembrane+Maurer&#8217;s+cleft+protein+family,+PfMC-2TM+and+the+130+kDa+Maurer&#8217;s+cleft+protein+define+different+domains+of+the+infected+erythrocyte+intramembranous+network.">Proteins of the Plasmodium falciparum. two transmembrane Maurer&#8217;s cleft protein family, PfMC-2TM and the 130 kDa Maurer&#8217;s cleft protein define different domains of the infected erythrocyte intramembranous network.</a> Parasitol Res. 104 (4), 875-891 (2009).</li><li>Tsuboi, T., 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=Wheat+germ+cell-free+system-based+production+of+malaria+proteins+for+discovery+of+novel+vaccine+candidates.">Wheat germ cell-free system-based production of malaria proteins for discovery of novel vaccine candidates.</a> Infect. Immun. 76 (4), 1702-1708 (2008).</li><li>Tsuboi, T., Takeo, S., Sawasaki, T., Torii, M., Endo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+approach+to+the+production+of+vaccines+against+the+malaria+parasite.">An efficient approach to the production of vaccines against the malaria parasite.</a> Methods in Mol. Biol. 607, 73-83 (2010).</li><li>Wang, 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+versatile+protein+microarray+platform+enabling+antibody+profiling+against+denatured+proteins.">A versatile protein microarray platform enabling antibody profiling against denatured proteins.</a> Proteomics Clin. Appl. 7 (5-6), 378-383 (2013).</li><li>Wang, T., Fujioka, H., Drazba, J. A., Sam-Yellowe, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhop-3+protein+conservation+among+Plasmodium.">Rhop-3 protein conservation among Plasmodium.</a> species and induced protection against lethal. 99 (3), 238-252 (2006).</li><li>Yadavalli, R., Ledger, C., Sam Yellowe, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+human+cell+free+expression+for+synthesis+of+malaria+proteins.">In vitro human cell free expression for synthesis of malaria proteins.</a> Parasitol Res. 111 (6), 2461-2465 (2012).</li><li>Machida, K., Masutan, M., Imataka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Synthesis+in+vitro.:+Cell-Free+Systems+Derived+from+Human+Cells.">Protein Synthesis in vitro.: Cell-Free Systems Derived from Human Cells.</a> Intech. , (2012).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantification+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li></ol>

We do not have any competing financial interests in this study.

The important steps for successful expression of proteins using two step in vitro human cell free expression system and purification are: 1) successful amplification and cloning of genes into pT7CFE-CHis plasmid vector; 2) transcribing RNA at 30oC; 3) translation of protein at 30 &#176;C in the presence of RNAse inhibitor; 4) developing the affinity based purification protocol using resin beads coated with Ni and 5) analyzing results on western blot using polyclonal and monoclonal antibodies. The important ste...

In malaria research, expression of immunogenic proteins using prokaryotic or eukaryotic cell based systems remains a challenge. The A-T richness of the Plasmodium genome and unknown post translational mechanisms14,7, contribute to the difficulties associated with obtaining properly folded and immunogenic proteins for antibody production and vaccine studies. Prokaryotic systems such as Escherichia coli have been used for recombinant protein expression. Prokaryotic systems are low cost, effective, produce high yields of recombinant protein and have multiple cloning vectors. Host E. coli cells are easy to transform and cells grow rapidly. However, prokaryotic systems have limitations such as lack of amino acid substitution, post-translational modification, risk of contamination, heterogeneous products and accumulation of recombinant proteins within inclusion bodies24.
In a study by Mehlin et al. (2006), 1000 open reading frames (ORF) were expressed. Approximately 7% of the expressed proteins were soluble14. The insolubility observed is due to the biased nature of the genes and the high frequency of codons that are used ideally by the Plasmodium A-T rich genome14. An alternative strategy to overcome this problem has been developed by using plasmids or host cells containing tRNAs that recognize rare codons or codons that match the frequencies3. Even after performing these optimizations, very small portions of proteins are expressed as soluble, active and immunogenic14. The cell-free expression systems contain all the components necessary for transcription and translation such as ribosomes, initiation factors, elongation factors (translations factors), tRNA and aminoacyl-tRNA synthetases. Both transcription and translation reactions are coupled in one-step procedure11,29. The transcription reaction is performed in a tube before appropriate amount of mRNA is incubated with translation machinery in a different tube11,29. Although these methods are successful in Plasmodium sp. protein expression, a major drawback is the length of time for protein expression, which is approximately 22 hr29. In addition, the high cost of supplies for inclusion in the protocols29, the labor-intensive preparations of the cell lysate and inconsistencies in component preparations, make these systems unattainable. The main focus of researchers for development of a cell free expression system depends on factors such as rapid genetic modification, fast yields with high concentrations and straight forward lysate preparations1.
Eukaryotic systems such as yeast, mammalian cell lines, baculovirus mediated expression system, Tetrahymena thermophilia, Dictyostelium discoideum and parasitic expression systems such as Leishmania have been used for recombinant protein expression7. Eukaryotic systems share phylogenetic relationship and therefore also share characteristics such as glycosylation, acylation, disulphide bond formation, chaperone interaction, proteolysis and sub-cellular compartmentalization. Protein secretion events in eukaryotes prevent accumulation and decreases toxicity for expression systems13. The use of yeast systems is preferred as they are suitable for protein expression in large scale and for obtaining high yields4. Two P. falciparum proteins PfCP-29 and Pvs25 were produced using the yeast system4. However, a major drawback in the synthesis of most of the proteins was irregular N and O-glycosylation patterns, improper folding and truncation of proteins from their native form4.
The use of mammalian cells for synthesis of recombinant proteins is labor-intensive and it is expensive to maintain stable recombinant cell lines4. Therefore, mammalian cells have been limited for the analysis of protein signaling, protein interactions and parasite-host interactions and also for testing DNA vaccines4. The Human DNA and mRNA in vitro protein expression cell-free system described herein is a HeLa cell-derived mammalian-based system that expresses proteins in 3 hr. Proteins expressed using pT7CFE1-cHis expression vector have a C-terminal tag facilitating identification and purification. The HeLa based system is supplemented with translation initiation factors and a translation regulator, thereby enhancing the efficiency of the translation system. Proteins can be rapidly translated, screened, verified and processed for use in various immunologic and structural applications15.
Eukaryotic cell-free expression systems such as wheat germ and rabbit reticulocyte are currently used for the expression of various eukaryotic proteins including Plasmodium proteins11,29. In addition, E. coli based cell-free systems are also employed for eukaryotic protein expression11. Table 1 summarizes different cell free expression systems by comparing features of the systems as well as the ease of using the systems for protein expression. The human cell-free system in comparison to the others has the ability to perform post and co-translational modifications and is less expensive. Codon optimization is possible and all the reactions can be performed in 3 hr. The HeLa system is an ideal translation system for protein synthesis in the laboratory setting.
A major advantage of human cell free expression systems over other cell free systems is the availability of several different cell lines derived from different organs and tissues. Several varieties of cell free systems can be designed depending on the cell line. The extracts derived from mammalian cells have higher efficiency to synthesize large proteins than any other cell free expression systems. These cell free expression systems can also be used in diagnostic and protein characterization applications such as micro arrays30. The popular HeLa cell lines are most widely used to carry out the expression of proteins33. The endoplasmic reticulum in the HeLa cell lines is underdeveloped, leading to the absence of post-translational glycosylation modification activity33. However, this system is believed to be advantageous for Plasmodium protein synthesis as Plasmodium also lacks glycosylation post translation modification29. The HeLa cell free transcription-translation protocol is easy to perform, inexpensive and proteins can be expressed in 90 min, ready for purification and further downstream applications.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Two-step in vitro human cell free expression system</td>
			<td>Thermo fischer</td>
			<td>88856</td>
			<td>Discontinued. Always store at -80oC. Do not thaw in warm water</td>
		</tr>
		<tr>
			<td>One-step in vitro coupled translation system</td>
			<td>Thermo fischer</td>
			<td>88860</td>
			<td>Always store at -80oC. Do not thaw in warm water</td>
		</tr>
		<tr>
			<td>ProBond Purification system</td>
			<td>Invitrogen</td>
			<td>K850-01</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>Probond Nickel-Chelating Resin</td>
			<td>Invitrogen</td>
			<td>R801-01</td>
			<td>Store at 4oC.</td>
		</tr>
		<tr>
			<td>&#160;A+ Human Blood</td>
			<td>Interstate Blood Bank</td>
			<td></td>
			<td>Store at 4oC.</td>
		</tr>
	</tbody>
</table>

hela based cell free expression systems for expression of plasmodium rhoptry proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

Validation of the expressed recombinant proteins through reactivity with specific antibodies is an important first step in confirming the proper folding of the expressed proteins. Recombinant malarial proteins were expressed using the one step and two step in vitro human cell free expression systems. The recombinant proteins are purified Ni-chelating affinity method. We then used antisera against whole merozoite rhoptries in western blotting of SDS-PAGE separated recombinant proteins.
...

Watch this Scientific Journal Video about HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins at JoVE.com

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

hela-based-cell-free-expression-systems-for-expression-plasmodium

Research

JoVE Journal

Biology

11.9K Views.  Cleveland State University. Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

Video: HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements1.
Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations2. Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer3. The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis4-6 and cell viability analysis with propidium iodide (PI)7-11.
Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population.
A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.

This protocol describes how to perform cell viability and fluorescence expression assays using the Tali Image-Based Cytometer.

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...