Applications of cell-free expressed protein microarrays Harini Chandra Affiliations Applications of cell-free expression-based protein microarrays have

Applications of cell-free expressed protein microarrays

Harini ChandraAffiliations

Applications of cell-free expression-based protein microarrays have seen a rampant increase over the

last decade due to the several advantages offered by these techniques over traditional cell-based

technologies.

Master Layout (Application 1)

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1 This animation consists of 3 parts:Application 1 - Biomarker detection

Application 2 - Immunological studiesApplication 3 - Protein interaction studies

Patient sera

p53 autoantibodies

Anti-human IgG

p53 positive sera

Transcription & translation

BSA, BS3 printing mix

cDNA

Cell-free lysate

Anti-GST antibodies

Anderson, K. A., Ramachandran, N., Wong, J., Raphael,J. V. et al., Application of protein microarrays for multiplexed detection of antibodies to tumor antigens in breast cancer. J. Proteome Res. 2008, 7, 1490–1499.

Protein microarray

Detection of p53 autoantibodies in human serum using cell-free expression based NAPPA microarrays.

Definitions of the components:Application 1 – biomarker detection

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11. cDNA: The complementary DNA expressing the antigen of interest along with the GST tag. This cDNA is transcribed and translated using suitable cell-free lysate. The expressed protein is then captured onto the microarray through the anti-GST antibody.

2. BSA, BS3 printing mix: The printing mix for generation of NAPPA protein microarrays consisted of bovine serum albumin (BSA), which significantly increased the binding efficiency of DNA, and BS3, which served as a protein cross-linker.

3. p53 autoantibodies: Antibodies against the tumour suppressor gene, p53, is believed to be one of the factors involved in breast cancer. Early detection of such biomarkers can have tremendous impact on disease treatment and monitoring prognosis. An increasing number of such biomarkers are being studied in order to facilitate the early identification of diseases.

4. Patient sera: Suitable patient sera samples were collected for the study. Sera was obtained from breast cancer patients, early stage ovarian cancer patients and healthy subjects.

5. Anti-human IgG: The binding of p53 autoantibody to the corresponding antigen is detected by means of anti-human IgG binding to the p53 autoantibody.

Definitions of the components:Application 1 – biomarker detection

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16. Protein microarray: The microscope slide on to which the DNA templates are immobilized and the entire expression is carried out. The target proteins bind to this array surface through an antibody.

7. p53 positive sera: Serum from those patients who have autoantibodies to p53 is termed as ‘p53 positive’. These patients will give positive detection signals when probed by the anti-human IgG.

Application 1, Step 1:

Action Audio Narration

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4Description of the action

Patient sera

p53 autoantibodies

Anti-human IgGTranscription

& translation

Cell-free lysate

cDNA

The light purple shapes must appear and bind to the blue V shaped objects.

First show the base with the circular strands and V-shaped objects bound to it. Then show the green cloud appearing followed by the arrow and the light purple objects. These must bind to the V-shaped objects. Then show the yellow cloud with maroon spade-shaped object. These must bind to the purple object followed by binding of green spade to the maroon spade.

The authors generated protein microarrays based on NAPPA expression, which they probed with diluted sera of breast cancer patients having p53 autoantibodies. Detection was carried out by means of HRP-linked anti-human IgG.



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The two rectangles must be shown as above.

First show the ‘p53 negative sera’ followed by the ‘p53 positive sera’ with the red spots.

This study detected p53 autoantibodies by means of NAPPA microarrays, which was confirmed by ELISA. The p53 levels were found to be directly related to tumour burden with serum antibody concentration decreasing after neoadjuvant chemotherapy.

p53 positive sera

p53 negative sera


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PCR amplified expression vectors

E. coli IVTTProteins expressed

Spotting

Patient sera

Experimentally exposed group

Naturally exposed group

Doolan, D. L., Mu, Y., Unal, B., Sundaresh, S. et al., Profiling humoral immune responses to P. falciparum infection with protein microarrays. Proteomics 2008, 8, 4680–4694.

Reaction tube

Use of cell-free expression based protein microarrays for detection of potential immunogenic proteins of Plasmodium falciparum.

This animation consists of 3 parts:Application 1 - Biomarker detection


Definitions of the components:Application 2 – immunological studies

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11. Reaction tube: Cell-free expression of the Plasmodium falciparum (Pf) proteins was carried out in this reaction tube.

2. PCR amplified expression vectors: Vectors encoding 250 putative Pf proteins were generated by PCR/recombination cloning. These were expressed using the E. coli IVTT.

3. E. coli IVTT: Proteins were expressed from the expression vectors using the E. coli cell-free in vitro transcription-translation (IVTT) mixture. The main drawbacks of this bacterial expression system include the absence of protein glycosylation and the lack of a proper redox environment to allow for correct protein folding and formation of disulphide bonds.

4. Proteins expressed: The 250 proteins were individually expressed with greater than 90% efficiency and printed without any further purification onto microarray slides.

Definitions of the components:Application 2 – immunological studies

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15. Patient sera: The printed microarray slides were then probed with serum samples from different patient groups:

a) Naturally exposed group: Group of (Kenyan) subjects that had been naturally exposed to Pf and had reported clinical symptoms within the previous year.

b) Experimentally exposed group: Volunteers who had never been infected were immunized with radiation attenuated Pf and then challenged with the bites of 5 infected mosquitoes. Samples were collected from these subjects prior to immunization, immediately prior to challenge and following challenge.



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The grey solution must be zoomed into and the figures on the right must be shown.

(Please redraw all images.)First show the grey solution in the tube which must be zoomed into. The figures on the right must then be shown followed by the curved arrow and then the coloured shapes. The figure below must then appear and the black shape must be shown to move horizontally from left to right.

The authors carried out cell-free expression of the PCR amplified vectors using an E. coli IVTT. They expressed 250 putative proteins that were printed directly onto microscopic array slides without any purification.

PCR amplified expression vectors

E. coli IVTT Proteins expressed

Array printingReaction tube

Protein expression and array printing



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The cloud figures must appear on the black parallelograms followed by the black rectangles with coloured spots.

First show the four black parallelograms with their labels followed by appearance of the cloud shaped figures on top of them. This is then followed by appearance of the black rectangles with coloured dots.

These arrays were probed with serum samples from patients who had been naturally exposed to Pf and who were experimentally exposed by means of radiation attenuated Pf. Authors successfully identified 72 highly immunoreactive protein antigens as well as 56 previously uncharacterized antigens that were serodominant, which can serve as potential vaccine targets.

Array probing and analysis

Patient sera

Experimentally exposed group

Naturally exposed group

Pre-immunization

Post-challenge

Anti-polyhistidine mouse Ab


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Ramachandran, N., Hainsworth, E., Bhullar, B., Eisenstein, S. et al., Self-assembling protein mircoarrays. Science 2004,305, 86–90.

Replication initiation DNAs

Cell-free lysate

Anti-GST antibodies

Expressed proteins

Query proteins

Protein interaction studiesProtein microarray

Identification of novel protein-protein interactions using NAPPA microarray.

This animation consists of 3 parts:Application 1 - Biomarker detection


Definitions of the components:Application 3 – Protein interaction studies

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11. Replication initiation DNAs: Human genes for 29 different proteins involved in the initiation of DNA replication were immobilized on to the array surface and expressed in duplicate using NAPPA.

2. Cell-free lysate: Rabbit reticulocyte lysate was applied as a single continuous layer across the slide and used for expression of the immobilized DNA.

3. Expressed proteins: The replication initiation proteins were expressed on the array surface where they became immobilized via the anti-GST antibodies. Signals were detected readily and were found to show high reproducibility between duplicates.

4. Protein interaction studies: Protein interaction studies were carried out between each of the 29 proteins expressed and a duplicate array of the same 29 proteins used as a query molecule, thereby generating a 29 X 29 interaction matrix. Such interaction studies can also be carried out with other protein or peptide molecules, as well as other biomolecules. These provide insights into pathway mapping, protein function determination and substrate identification.

5. Query protein: The protein molecule that is used to probe the protein array of interest in order to detect any possible interaction is known as the query protein.



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One of the spots on the array below must be zoomed into and the events drawn above must be shown.

First show the gray array surface below followed by one of the spots being zoomed into and appearance of the figures shown above. Show the base with the circular strands and green spade shaped objects bound to it. Then show the violet cloud appearing followed by the arrow and the purple and blue objects. These must bind to the spade-shaped objects.

Ramachandran et al., tested the use of NAPPA microarrays by immobilizing 29 sequence-verified human genes involved in replication initiation on the array surface and then expressing them in duplicate with RRL. The expressed proteins bound to the anti-GST antibodies present on the array surface.

Protein expression using NAPPA

Replication initiation DNAs

Cell-free lysate

Anti-GST antibodies

Expressed proteins

Protein microarray



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The coloured ‘query proteins’ must probe the grey array surface below.

First show the grey array surface with spots followed by the binding of the coloured proteins to this surface. Once these move about and finally bind to the grey surface, the figure on the right must appear.

The authors made use of each of these expressed proteins to probe another duplicate array of the same 29 proteins thereby generating a 29 x 29 protein interaction matrix. 110 interactions were detected between proteins of the replication initiation complex, of which 63 were previously undetected ones.

Protein interaction studies

Query proteins

Array with expressed immobilized target proteins

29 x 29 protein interaction matrix

Interactivity option 1:Step No: 1

Boundary/limitsInteracativity Type Options Results

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Match the following applications to their appropriate studies to understand more about them.

1. Biomarker discovery

2. Immunological studies

3. Protein interaction studies c) Rapid bead-based assay for multiplexed detection of antibodies to EBNA-1 and p53.

a) Identification of serodiagnostic tests and vaccine development against Coxiella burnetti.

b) High density NAPPA array approach for studying well characterized gene pairs

Match the following – drag and drop.

User should be allowed to drag and drop the options on the left onto the options in the right. Once the correct match is made, user is redirected to steps 2, 3 & 4.

Correct answer is 1-c, 2-a and 3-b. Once the user gets this correct match, he is redirected to steps 2, 3 and 4 to view the animations shown there.

Interactivity option 1:Step No: 2(a)1

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Biomarker discovery – rapid bead-based assay for multiplexed detection of antibodies to EBNA-1 and p53.

Anti-GST Abs coupled to beads

Luminex beads of different fluorescence

Cell-free expression of GST-tagged DNA

Expressed protein captured onto beads

Mixing

Antigen coated beads

Wong, J., Sibani, S., Lokko, N. N., LaBaer, J., Anderson, K. S., Rapid detection of antibodies in sera using multiplexed self-assembling bead arrays. J. Immunol. Methods 2009, 350, 171–182.

Action Audio NarrationDescription of the actionAs shown in animation.

First show the three cans on left top with the colored figures. Next show the cans below with the colored strands. These cans must be mixed together according to their color to give rise to the three cans in the middle panel. Again these three must be mixed to give the can on the right.

The authors developed a programmable multiplexed immunoassay where tagged antigens were expressed using in vitro transcription and translation and captured onto anti-tag coated beads. The synthesized proteins were immobilized on to the beads through the capturing agent. These beads were then mixed together.

Interactivity option 1:Step No: 2(b)1

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Biomarker discovery – rapid bead-based assay for multiplexed detection of antibodies to EBNA-1 and p53.

IgG-containing human serum

Enzyme linked secondary Abs


Substrate

Colored product detected


Show the yellow solution containing inverted Y shaped objects moving into the can and binding to the green shapes. Next, show the green inverted Y which must bind to the black inverted Y. Next the grey ‘sun’ shaped object must appear which must bind to the brown part of the ‘Ab’ and once binding occurs, it must turn bright green as shown in animation.

Serum was added to these coupled beads and human IgG detected by probing with enzyme-linked anti-human IgG. Colored reaction was observed on addition of substrate to the enzyme. The authors demonstrated this approach for detection of antibodies to Epstein-Barr virus nuclear antigen 1 (EBNA1) and p53.

Interactivity option 1:Step No: 3(a)1

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Immunological studies – identification of immunogens of Q-fever-causing Coxiella burnetti.

1988 PCR amplified ORFs

E. coli IVTT Proteins expressed

Array printing with crude IVTT lysate

96 well reaction plate

Nitrocellulose microarrays

Beare, P. A., Chen, C., Bouman, T., Pablo, J. et al., Candidate antigens for Q fever serodiagnosis revealed by immunoscreening of a Coxiella burnetii protein microarray. Clin. Vaccine Immunol. 2008, 15, 1771–1779.


(Please redraw all images.)Show the black plate on the left. One of the spots must be zoomed into and the next figure must appear which must then give rise to the ‘proteins’. These must be shown to move onto the array surface and the robotic arm must move across the surface.

Beare et al., carried out in vitro transcription and translation of 1988 ORFs of C. burnetti using E. coli based cell-free systems. 75% of the ORFs were successfully generated as full-length proteins and spotted onto nitrocellulose arrays.

Interactivity option 1:Step No: 3 (b)1

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Immunological studies – identification of immunogens of Q-fever-causing Coxiella burnetti.

Sera from acute Q-fever patients

Sera from vaccinated patients

75% of the ORFs generated full length proteins successfully. 50 C. burnetti proteins were found to react strongly with immune sera.



Show the grey parallelograms above which must then be covered with two solutions of slightly different color. Once this happens the array signals shown below must be displayed.

The arrays were probed with sera from patients who had been vaccinated as well as acute Q-fever patients. 50 proteins were identified that were found to react strongly with the immune sera.

Interactivity option 1:Step No: 4 (a)1

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Protein interaction studies - high density NAPPA array approach for studying well characterized gene pairs

647 unique genes printed on array

Cell-free lysate

Anti-GST antibodies

Expressed proteins

High density protein microarray

cDNA of query protein without GST tag

Unbound query proteins

Ramachandran, N., Raphael, J. V., Hainsworth, E., Demirkan,G. et al., Next-generation high-density self-assembling functional protein arrays. Nat. Methods 2008, 5, 535–538.


Show the grey surface with spots below. One spot must be zoomed into and the surface above must be show with the Y shaped objects and concentric circles bound to it. Unbound concentric circles must also appear along with the violet cloud. This must be shown to give rise to ‘proteins’ as shown.

The authors made use of high density NAPPA arrays to study protein interactions. 647 unique genes were printed on to the array surface and expressed by adding the cell-free lysate. cDNA of the query protein was also added to the same mixture such that the query was co-expressed but remained unbound due to the lack of a tag capturing agent.

Interactivity option 1:Step No: 4 (b)1

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Protein interaction studies - high density NAPPA array approach for studying well characterized gene pairs

Protein interaction studies

Antibodies to detect protein interactions

Protein interactions using Jun, Fos and MDM2 as queries detected


Action Audio NarrationDescription of the actionShow colored proteins binding to the grey surface followed by addition of the inverted Y shaped ‘antibodies’ which must bind to the colored proteins. This binding must generate the signal shown on the right.

The arrays were then probed with antibodies specific to the query protein. Authors detected various protein interactions using well known query proteins.

As shown in animation.

Questionnaire1

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1. Which of the following provides improved DNA binding to the microarray during NAPPA?

Answers: a) BS3 b) GST c) BSA d) Avidin

2. Which of these expression systems will not allow for protein glycosylation?

Answers: a) Rabbit Reticulocyte Lysate b) E. coli S30 c) Wheat germ extract d) All of the

above

3. How many immunoreactive protein antigens of P. falciparum were detected in the study by

Doolan et al.?

Answers: a) 20 b) 45 c) 250 d) 72

4. How many previously uncharacterized protein interactions were detected by Ramachandran

et al. (2004) in their 29 x 29 interaction matrix?

Answers: a) 110 b) 56 c) 37 d) 63

5. C. burnetti causes which of the following infections?

Answers: a) Breast cancer b) Ovarian cancer c) Malaria d) Q-fever

Links for further readingBooks:

New and Emerging Proteomic Techniques. Edited by Dobrin Nedelkov & Randall W.Nelson

(Humana Press).

Research papers: Chandra, H. & Srivastava, S. Cell-free synthesis-based protein microarrays and their applications.

Proteomics 2010, 10, 1-14.

Davies, D. H., Liang, X., Hernandez, J. E., Randall, A. et al., Profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine anddiagnostic antigen discovery. Proc. Natl. Acad. Sci. USA 2005, 102, 547–552.

Eyles, J. E., Unal, B., Hartley, M. G., Newstead, S. L. et al., Immunodominant Francisella tularensis antigens identified using proteome microarray. Proteomics 2007, 7, 2172–2183.

Lopez, J. E., Beare, P. A., Heinzen, R. A., Norimine, J. et al., High-throughput identification of T-lymphocyte antigens from Anaplasma marginale expressed using in vitro transcription and translation. J. Immunol. Methods 2008, 332, 129–141.

Anderson, K. A., Ramachandran, N., Wong, J., Raphael,J. V. et al., Application of protein microarrays for multiplexed detection of antibodies to tumor antigens in breast cancer. J. Proteome Res. 2008, 7, 1490–1499.

Links for further readingResearch papers:

Doolan, D. L., Mu, Y., Unal, B., Sundaresh, S. et al., Profiling humoral immune responses to P. falciparum

infection with protein microarrays. Proteomics 2008, 8, 4680–4694.

Ramachandran, N., Hainsworth, E., Bhullar, B., Eisenstein, S. et al., Self-assembling protein mircoarrays. Science 2004,305, 86–90.




Documents

Applications of cell-free expressed protein microarrays Harini Chandra Affiliations Applications of cell-free expression-based protein microarrays have