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>

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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.
...

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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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.0K 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

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

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

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.

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

Biochemistry

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Immunology and Infection

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

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

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

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

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

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

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

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

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

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

Streamlined Protocol for Leishmania Translational Extract (LTE) Production

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

This protocol outlines the production and optimization of a eukaryotic Cell-Free Protein Expression System (CFPS) derived from the unicellular flagellate Leishmania tarentolae, referred to as Leishmania Translational Extract or LTE. Although this organism originally evolved as a parasite of geckos, it can be cultivated easily and inexpensively in flasks or bioreactors. Unlike Leishmania major, it is non-pathogenic to humans and does not require special laboratory precautions. Another advantage of using Leishmania for CFPS is that the addition of a single antisense oligonucleotide to the CFPS, targeting a conserved splice leader sequence on the 5'-end of all protein-coding RNAs, can suppress endogenous protein expression. We provide procedures for cell disruption and lysate processing, which have been simplified and improved compared to previous versions. These procedures start with simple flask cultures. Additionally, we explain how to introduce genetic information using vectors containing species-independent translation initiation sites (SITS) and how to perform straightforward batch optimization and quality control to ensure consistent protein expression quality.

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.

This protocol outlines the production and optimization of a eukaryotic Cell-Free Protein Expression System (CFPS) derived from the unicellular flagellate ...

Bioengineering

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

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

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

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

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

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

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.

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

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

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