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

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.