Name: HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins (Video) | JoVE Journal
Uploaded: 2015-06-10T14:00:00.000+00:00
Duration: 9 min 3 s
Description: Learn to express Plasmodium rhoptry proteins using HeLa-based cell-free systems with one-step and two-step in vitro translation methods. This technique enables rapid protein production and affinity purification for malaria research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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/pubmed?term=((Leishmania.+cell-free+expression+system%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">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/pubmed?term=((Towards+the+construction+of+expressed+proteomes+using+a+Leishmania+tarentolae.+based+cell-free+expression+system%5BTitle%5D)+AND+%22PLOS+One%22%5BJournal%5D)">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/pubmed?term=((Overcoming+codon+bias%3A+A+method+for+high-level+over-expression+of+Plasmodium.+and+other+AT-rich+parasite+genes+in+Escherichia+coli%5BTitle%5D)+AND+%22Int+J+Parasitol%22%5BJournal%5D)">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/pubmed?term=((Heterologous+expression+of+plasmodial+proteins+for+structural+studies+and+functional+annotation%5BTitle%5D)+AND+%22Malar.+J%22%5BJournal%5D)">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/pubmed?term=((Profiling+humoral+immune+responses+to+P.+falciparum.+infection+with+protein+microarrays%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">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/pubmed?term=((Cell-free+expression+systems+for+eukaryotic+protein+production%5BTitle%5D)+AND+%22Curr.+Opin.+Biotechnol%22%5BJournal%5D)">Cell-free expression systems for eukaryotic protein production.</a> Curr. Opin. Biotechnol. 17 (4), 373-380 (2006).</li>
<li>Fernández-Robledo, J. A., Vasta, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+recombinant+proteins+from+protozoan+parasites%5BTitle%5D)+AND+%22Trends+Parasitol%22%5BJournal%5D)">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/pubmed?term=((Protein+glycosylation+in+the+malaria+parasite%5BTitle%5D)+AND+%22Parasitol.+Today%22%5BJournal%5D)">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/pubmed?term=((Human+protein+factory+for+converting+the+transcriptome+into+an+in+vitro-expressed+proteome%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHinnebusch%2C+A+%22Mechanism+and+regulation+of+initiator+methionyl-tRNA+binding+to+ribosomes%22">Mechanism and regulation of initiator methionyl-tRNA binding to ribosomes</a>. 5, Cold Spring Harbor Laboratory. Cold Spring Harbor, N.W. 185-243 (2000).</li>
<li>Hino, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficiency+of+cell-free+protein+synthesis+based+on+a+crude+cell+extract+from+Escherichia+coli.%2C+wheat+germ%2C+and+rabbit+reticulocytes%5BTitle%5D)+AND+%22Journal+of+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Discovery+of+gene+function+by+expression+profiling+of+the+malaria+parasite+life+cycle%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Strategies+for+achieving+high-level+expression+of+genes+in+Escherichia+coli%5BTitle%5D)+AND+%22Microbiol.+Rev%22%5BJournal%5D)">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/pubmed?term=((Heterologous+expression+of+proteins+from+Plasmodium+falciparum.%3A+results+from+1000+genes%5BTitle%5D)+AND+%22Mol.+Biochem.+Parasitol%22%5BJournal%5D)">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/pubmed?term=((A+human+cell-derived+in+vitro+coupled+transcription%2Ftranslation+system+optimized+for+production+of+recombinant+proteins%5BTitle%5D)+AND+%22Protein+Expr.+Purif%22%5BJournal%5D)">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/pubmed?term=((Proteome+analysis+of+rhoptry%3A+Enriched+isolated+from+Plasmodium.+merozoites%5BTitle%5D)+AND+%22Journal+of+Proteome+Research%22%5BJournal%5D)">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/pubmed?term=((Plasmodium+falciparum.+protein+located+in+Maurer%E2%80%99s+clefts+underneath+knobs+and+protein+localization+in+association+with+Rhop-3+and+SERA+in+the+intracellular+network+of+infected+erythrocytes%5BTitle%5D)+AND+%22Parasitol+Res%22%5BJournal%5D)">Plasmodium falciparum. protein located in Maurer’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/pubmed?term=((A+Plasmodium.+gene+family+encoding+Maurer%E2%80%99s+cleft+membrane+proteins%3A+Structural+properties+and+expression+profiling%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">A Plasmodium. gene family encoding Maurer’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/pubmed?term=((Plasmodium+yoelii.%3A+Novel+rhoptry+proteins+identified+within+the+body+of+merozoite+rhoptries+in+rodent+Plasmodium+malaria%5BTitle%5D)+AND+%22Exp+Parasitol%22%5BJournal%5D)">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/pubmed?term=((Isolation+of+merozoite+rhoptries%2C+identification+of+novel+rhoptry+associated+proteins+from+P.+yoelii.%2C+P.+chabaudi.%2C+P.+berghei.+and+conserved+interspecies+reactivity+of+organelles+and+proteins+with+P.+falciparum.+rhoptry-specific+antibodies%5BTitle%5D)+AND+%22Exp+Parasitol%22%5BJournal%5D)">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/pubmed?term=((Plasmodium+falciparum.+rhoptry+proteins+of+140%2F130%2F110kd+%28Rhop-H%29+are+located+in+an+electron+lucent+compartment+in+the+neck+of+the+rhoptries%5BTitle%5D)+AND+%22J+Euk+Microbial%22%5BJournal%5D)">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/pubmed?term=((Molecular+Organization+and+cross+linking+analysis+of+the+Plasmodium+falciparum+erythrocyte+binding+proteins+Rhop-H+and+SERA%5BTitle%5D)+AND+%22J+Protozool.+Res%22%5BJournal%5D)">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/pubmed?term=((Secretion+of+Plasmodium+falciparum.+rhoptry+protein+into+plasma+membrane+of+host+erythrocytes%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((Identification+of+humoral+immune+responses+in+protein+microarrays+using+DNA+microarray+data+analysis+techniques%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">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/pubmed?term=((Overview+of+bacterial+expression+systems+for+heterologous+protein+production%3A+from+molecular+and+biochemical+fundamentals+to+commercial+systems%5BTitle%5D)+AND+%22Appl.+Microbiol.+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((Human+malaria+parasites+in+continuous+culture%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Proteins+of+the+Plasmodium+falciparum.+two+transmembrane+Maurer%E2%80%99s+cleft+protein+family%2C+PfMC-2TM+and+the+130+kDa+Maurer%E2%80%99s+cleft+protein+define+different+domains+of+the+infected+erythrocyte+intramembranous+network%5BTitle%5D)+AND+%22Parasitol+Res%22%5BJournal%5D)">Proteins of the Plasmodium falciparum. two transmembrane Maurer’s cleft protein family, PfMC-2TM and the 130 kDa Maurer’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/pubmed?term=((Wheat+germ+cell-free+system-based+production+of+malaria+proteins+for+discovery+of+novel+vaccine+candidates%5BTitle%5D)+AND+%22Infect.+Immun%22%5BJournal%5D)">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/pubmed?term=((An+efficient+approach+to+the+production+of+vaccines+against+the+malaria+parasite%5BTitle%5D)+AND+%22Methods+in+Mol.+Biol%22%5BJournal%5D)">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/pubmed?term=((A+versatile+protein+microarray+platform+enabling+antibody+profiling+against+denatured+proteins%5BTitle%5D)+AND+%22Proteomics+Clin.+Appl%22%5BJournal%5D)">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/pubmed?term=((Rhop-3+protein+conservation+among+Plasmodium%5BTitle%5D)+AND+%22species+and+induced+protection+against+lethal%22%5BJournal%5D)">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/pubmed?term=((In+vitro+human+cell+free+expression+for+synthesis+of+malaria+proteins%5BTitle%5D)+AND+%22Parasitol+Res%22%5BJournal%5D)">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/pubmed?term=((Protein+Synthesis+in+vitro.%3A+Cell-Free+Systems+Derived+from+Human+Cells%5BTitle%5D)+AND+%22Intech%22%5BJournal%5D)">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/pubmed?term=((A+rapid+and+sensitive+method+for+the+quantification+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">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><tbody><tr><td>2-step &lt;em&gt;in vitro&lt;/em&gt; human cell free expression system</td><td>Thermo Fisher</td><td>88856</td><td>Discontinued. Always store at -80 &amp;deg;C. Do not thaw in warm water</td></tr><tr><td>1-step &lt;em&gt;in vitro&lt;/em&gt; coupled translation system</td><td>Thermo fischer</td><td>88860</td><td>Always store at -80 &amp;deg;C. Do not thaw in warm water</td></tr><tr><td>ProBond Purification system</td><td>Invitrogen</td><td>K850-01</td><td>Store at RT</td></tr><tr><td>Probond Nickel-Chelating Resin</td><td>Invitrogen</td><td>R801-01</td><td>Store at 4&lt;sup&gt;o&lt;/sup&gt;C.</td></tr><tr><td>A+ Human Blood</td><td>Interstate Blood Bank</td><td></td><td>Store at 4 &amp;deg;C.</td></tr></tbody></table>

hela based cell free expression systems for expression of plasmodium 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 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.
...

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.

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 overall goal of the following experiment is to translate plasmodium proteins using hela based cell-free expression systems and to purify the proteins from micro volumes of translated product. This is achieved by cloning plasmodium genes to prepare recombinant plasmids for two step and one step in vitro translation, cell-free expression of malaria recombinant RT tree proteins. To translate the recombinant proteins, the prepared plasmid is incubated with transcription mix.When using the two step expression system, which transcribes mRNA, the resulting mRNA is then added to the translation mix for protein translation. While using the one step expression system, the recombinant plasmid is incubated with hela cell lysate for transcription and translation of recombinant proteins. Recombinant proteins are then purified from micro volumes of translation product.The results show successful expression and purification based on western blotting analysis. The main advantage of this technique over others, such as the wheat germ expression system and the rabbit reticulocyte lysate system is that this system can express proteins in three hours. No coding optimization is required.It is affordable and easy to use. Multiple antigens can be screened on microarrays making the detection of antigens for therapy and diagnosis possible. Generally, individuals new to this method will struggle because it is cell-free expression, which expresses proteins in micro volumes making protein purification extremely difficult.We first had an idea to use this system as we could not explo express proteins using e coli and found other expression systems too expensive and time consuming. We decided to explore hela based cell-free expression system as an alternate system. Prepare 20 microliters of transcription mixture containing recombinant plasmid vector DNA as described in the text protocol for two-step human in vitro protein expression.Using DNA templates. Mix the components in a micro centrifuge tube and incubate for 75 minutes at 30 degrees Celsius in a water bath. Then take two microliters of the transcription mixture and add it to 23 microliters of translation mixture containing hela cell lysate.Incubate the resulting mixture at 30 degrees Celsius for 90 minutes. Store the translated products at minus 20 degrees Celsius. Next, prepare 25 microliters of the transcription and translation mixture containing the recombinant plasmid vector, DNA and he a lysate as described in the text protocol for one step human coupled in vitro translation protein expression for DNA templates.Incubate this reaction mixture for 90 minutes to six hours at 30 degrees Celsius and store the translation products at minus 20 degrees Celsius. To purify the expressed recombinant proteins, add 75 microliters of one X purification buffer to 25 microliters of translation product to make 100 microliters of purification mixture. Wash the nickel resin twice by adding 300 microliters of distilled water and 300 microliters of the binding buffer.Resuspend the resin and centrifuge at 14, 000 Gs for one minute to remove excess ethanol. Next, add 100 microliters of the prepared purification solution to 100 microliters of nickel chelating resin, and incubate the mixture on an end. End shaker for 60 minutes at room temperature.Centrifuge the mixture at 14, 000 Gs for one minute and collect the supernat into a fresh tube labeled as flow through. Wash the resin with 100 microliters of one x wash buffer, then centrifuge at 14, 000 GS for one minute and collect the super named in a fresh tube labeled wash. Repeat this step twice following the wash.Add 100 microliters of one x elution buffer to the resin and incubate on an end end shaker for 15 minutes. Then centrifuge at 14, 000 Gs for one minute. Perform the Ellucian step twice each time.Collect the supernatant into a fresh micro centrifuge tube and label as eluate after ellucian. Wash the resin twice as before and then store it at four degrees Celsius. Store the flow through washes and elution samples at minus 20 degrees Celsius or lower to perform western blotting.First, prepare separate tubes of schon extracts from P falsy parum e coli, expressed recombinant R three proteins, the prepared recombinant proteins and the protein products purified using the affinity method. Next, add electrophoresis sample buffer containing mercaptoethanol to each tube for a final volume of 20 microliters. To make solubilized protein samples boil the tubes for two minutes.Load the solubilized protein samples onto 10%SDS page gels to separate the proteins after running the gels. Transfer the separated proteins from the SDS page gels to nitrocellulose paper by electrophoresis at 35 milliamps of current per gel in a semi dry western blotting chamber for two hours following transfer and blocking of the nitrocellulose paper with 2%non-fat milk. Incubate the nitrocellulose paper with the polyclonal antibodies listed in the text protocol for negative controls.Also incubate nitrocellulose paper in one to 100 diluted normal mouse and rabbit serum and spent culture super named from SB two myeloma cells. Following incubation of the nitrocellulose paper at four degrees Celsius overnight washed four times with blot buffer and incubate with species specific secondary antibodies, conjugated to horse radish peroxidase, diluted one to 1000 in 2%milk. Then wash the nitrocellulose paper again four times with blot buffer.Finally incubate the washed nitrocellulose paper with a color development solution, A plus B for 30 minutes in the dark, successful expression of plasmodium proteins using in vitro human cell-free expression systems was confirmed by RT tree specific rabbit antier as shown previously expressed recombinant proteins were not recognized by antisera against parasitosis vae proteins from p FALs parem and P Yoi Lee, demonstrating the specificity of rabbit antisera 6 76 against the expressed proteins. Normal rabbit serum did not react with recombinant proteins. Translated using the two-step in vitro human cell-free expression system and the one step coupled in vitro translation system.Successful purification of translated proteins from 25 microliters of translated protein products is shown here. Specifically successful purification of mara's cleft transmembrane protein is shown. Successful purification of a hypothetical protein from plasmodium RT tree protein encoding genes is also shown.Finally, successful purification of armadillo repeats containing plasmodium RT tree protein is shown. The yield of protein after purification is 3.5 micrograms per 25 microliters After its development. This technique paved way for researchers in the field of parasitology for protein characterization in parasites, protein-protein interactions, antibody inhibitory studies, and vaccine development in the parasitology field.After watching this video, you should understand how to use the healer cell-free based cell expression system to express plasmodium proteins in three hours. You will also have an understanding of how to purify the proteins from micro volumes of translated product.

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

Research

JoVE Journal

Biology

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

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.

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

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

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

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

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

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

Leishmania Translational Extract (LTE) is a eukaryotic cell-free protein expression system derived from the single-celled parasite, Leishmania tarentolae. This optimized protocol makes LTE simple and cost-effective to manufacture. It is suitable for various applications focused on the multiparallel expression and study of complex eukaryotic proteins and their interactions.

Production and Optimization of LTE, a Leishmania tarentolae Derived Cell-Free Protein Expression System for Recombinant Protein Production

Chemical genetics involves the substitution of a gatekeeper residue with an amino acid containing a different side chain at the target locus. Here, we have generated a mutant parasite containing a hypomorphic allele of cdpk1 and identified compensatory pathways adopted by the parasite in the mutant background.

Understanding the Development of Compensatory Pathways in a Mutant Malaria Parasite Harbouring Hypomorphic Allele of Plant-Like Kinases

Learn to express Plasmodium rhoptry proteins using HeLa-based cell-free systems with one-step and two-step in vitro translation methods. This technique enables rapid protein production and affinity purification for malaria research.

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

Explore More Videos