1. Cell-free expression based protein microarrays Harini Chandra Affiliations Cell-free expression involves the rapid, in situ synthesis of proteins from their corresponding DNA templates directly on the microarray surface. Protein arrays generated by this technique have shown great potential in eliminating the drawbacks of traditional cell- based methods.

2. Master Layout (Part 1) 5 3 2 4 1 This animation consists of 5 parts: Part 1 – Protein in situ array (PISA) Part 2 – Nucleic Acid Programmabe Protein Array (NAPPA) Part 3 – Multiple Spotting Technique (MIST) Part 4 – DNA Array to Protein Array (DAPA) Part 5 – HaloTag technique Transcription Translation PCR generated DNA construct Ribosomes mRNA Tagged protein Tag-capturing agent Cell-free lysate He, M., Taussig, M. J., Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method). Nucleic Acids Res. 2001, 29, e73.

3. Definitions of the components: Part 1 – Protein in situ array (PISA) 5 3 2 4 1 1. PCR generated DNA construct: The DNA construct used for protein expression is generated by PCR and encodes the protein of interest, a T7 promoter, sequences for initiation of translation, suitable termination sequences as well as an N- or C-terminal tag sequence for immobilization onto the microarray surface. 2. Cell-free lysate: Crude cell lysates obtained from growing cells contain all the essential machinery that are required for transcription and translation to take place. Nucleotides, essential amino acids and other energy- generating factors are added exogenously. Commonly used expression systems include E. coli S30, wheat germ extract (WGE) and rabbit reticulocyte lysate (RRL). 3. Transcription: The process by which mRNA is synthesized from its corresponding DNA template with the help of suitable enzymes and factors. 4. mRNA: The messenger RNA (mRNA) is transcribed from a DNA template and contains the coding information for the corresponding protein product. 5. Ribosomes: These are the site of synthesis of proteins from their respective mRNA. They are made up of a large and small subunit and carry out translation with the help of tRNAs, enzymes and other elongation and termination factors.

4. Definitions of the components: Part 1 – Protein in situ array (PISA) 5 3 2 4 1 6. Translation: The process by which the mRNA code is converted into its protein sequence. The mRNA is read in the form of three letter codes known as codons which specify one amino acid. 7. Tagged protein: The protein synthesized by translation has a specific tag molecule that allows the protein to get immobilized onto the array surface as soon as it is produced. (eg. His 6 tag). 8. Tag-capturing agent: This is a specific molecule that has been coated onto the array surface so that the protein obtained by cell-free synthesis will get captured onto the array due to the specific binding interaction between the tag and tag-capturing agent. (eg. Nickel-nitrilo triacetic acid, Ni-NTA).

5. Part 1, Step 1: ActionAudio Narration 1 5 3 2 4 Description of the action Tag-capturing agent Protein microarray Array surface One of the spots on the protein microarray must be zoomed into and the rest must be shown. First show the parallelogram with dots as shown. Zoom into one of the dots and show the figure drawn above. In PISA, the protein microarray surface is coated with a suitable tag-capturing agent that can immobilize the protein of interest through specific interactions once it is produced.

6. Part 1, Step 2: ActionAudio Narration 1 5 3 2 4 Description of the action Transcription Translation PCR generated DNA construct Ribosomes mRNA Tagged protein Tag-capturing agent RNA Polymerase (part of cell-free lysate) As shown in the animation First show the yellow strands followed by the green oval moving along them and then the appearance of the green strands. Next show the blue round structures moving along the green strands and appearance of the blue protein shapes. This is followed by the surface at the bottom with the moon shape. The oval must move down and get attached to this as shown. The protein is expressed from its corresponding DNA using cell-free lysates such as E.coli S30 or rabbit reticulocyte lysate (RRL). The tagged protein is then captured specifically onto the array surface through the tag-capturing agent. PISA successfully overcame drawbacks of cell-based techniques such as protein insolubility, aggregation etc.

7. Master Layout (Part 2) 5 3 2 4 1 This animation consists of 5 parts: Part 1 – Protein in situ array (PISA) Part 2 – Nucleic Acid Programmable Protein Array (NAPPA) Part 3 – Multiple Spotting Technique (MIST) Part 4 – DNA Array to Protein Array (DAPA) Part 5 – HaloTag technique Transcription Translation cDNA +GST tag Anti-GST antibodies GST tag Cell-free lysate Aminosilane coated glass slide 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. GST BSA BS 3

8. Definitions of the components: Part 2 – Nucleic acid programmable protein array (NAPPA) 5 3 2 4 1 1. Aminosilane coated glass slide: A glass slide coated with aminosilane reagent is used for cell-free protein expression in NAPPA. 2. cDNA+GST tag: High quality cDNA containing the gene to be expressed as its glutathione-S-transferase (GST) fusion. This is immobilized onto the array surface. 3. BSA: The protein bovine serum albumin (BSA) is added as part of the master-mix. This improves the binding efficiency of the cDNA onto the array surface. 4. BS 3 : BS 3 is a cross-linking agent that facilitates immobilization of the capture antibody on to the array surface. BS 3 is also added as part of the NAPPA master-mix. 5. GST tag: The protein obtained from translation of the mRNA is generated as a GST fusion i.e. it has a GST tag that can specifically bind onto the microarray surface through the antibody immobilized on the surface. 6. Anti-GST antibodies: Antibodies that bind specifically to the GST tag on the synthesized protein are immobilized on the surface of the array thereby allowing the expressed protein to be co-localized with its corresponding DNA. This technique therefore does not produce pure protein arrays but is capable of generating very high density microarrays.

9. Part 2, Step 1: ActionAudio Narration 1 5 3 2 4 Description of the action Protein microarray Aminosilane coated glass slide One of the spots on the protein microarray must be zoomed into and the figure on topmust be shown. First show the parallelogram with dots as shown. Next the hand must move across the surface and then disappear with gradual appearance of the pink solution on top. Next, zoom into one of the dots and show the figure drawn above. An aminosilane-coated glass slide forms the array surface for NAPPA. To this, the NAPPA master mix is added which consists of BSA, BS 3, GST-tagged cDNA and the anti-GST capture antibodies. The BSA improves efficiency of immobilization of the cDNA onto the array surface while the BS 3 cross-linker facilitates binding of the capture antibody. Anti-GST antibody BSA BS 3 GST NAPPA master mix cDNA + GST tag

10. Part 2, Step 2: ActionAudio Narration 1 5 3 2 4 Description of the action As shown in the animatio n. First show the surface at the bottom bound to the green Y shaped objects and yellow strands. Then the pink circles must appear and move up as shown followed by the arrow and the purple strands. The green shape must appear on top of the purple strand and move across the strand as shown. The yellow shape with the triangle then appears and this must move in such a way that it binds to the green shapes below. The cDNA is expressed using a cell-free extract to give the corresponding protein with its GST tag fused to it. This tag enables capture of the protein onto the slide by means of anti-GST antibodies. NAPPA technique can generate very high density arrays but the protein remains co-localized with the cDNA. Transcription Translation cDNA + GST tag Anti-GST antibodies GST tag RNA Polymerase (part of cell-free lysate) mRNA Ribosomes GST BSA BS 3

11. Master Layout (Part 3) 5 3 2 4 1 This animation consists of 5 parts: Part 1 – Protein in situ array (PISA) Part 2 – Nucleic Acid Programmabe Protein Array (NAPPA) Part 3 – Multiple Spotting Technique (MIST) Part 4 – DNA Array to Protein Array (DAPA) Part 5 – HaloTag technique Cell-free lysate First spotting Second spotting DNA template Expressed protein Tagged detection antibody Angenendt, P., Kreutzberger, J., Glokler, J., Hoheisel, J. D., Generation of high density protein microarrays by cell-free in situ expression of unpurified PCR products. Mol. Cell.Proteomics 2006, 5, 1658–1666.

12. Definitions of the components: Part 3 – Multiple spotting technique (MIST) 5 3 2 4 1 1. First spotting: This step involves the spotting of DNA template coding for the protein of interest in as little as fg quantities, onto the solid array surface. 2. DNA template: The DNA that codes for the protein of interest and is expressed by transcription and translation. 3. Second spotting: After the template DNA is spotted on to the array surface, the cell-free lysate is then transferred exactly on top of the first spot. This second spotting step marks the beginning of expression of the template DNA. 4. Expressed protein: The proteins produced by cell-free expression from the corresponding DNA templates are immobilized on the array surface either through a tag-capturing agent or more commonly, by means of non-specific interactions. These proteins can then be detected by suitably tagged antibodies. 5. Tagged detection antibody: Antibodies specific to the protein of interest can be added to the array surface for detection. The fluorescent tag molecules will indicate protein expression upon specific binding interactions between the protein and antibody.

13. Part 3, Step 1: ActionAudio Narration 1 5 3 2 4 Description of the action As shown in the animatio n. First show the parallelogram with dots. Then show the hand moving down into one of the spots followed by appearance of text ‘first spotting’ and then disappearance of hand and text. That spot must then be zoomed into and the figure on top must be shown. The first spotting step of the multiple spotting technique, which is also capable of producing high density arrays, involves the addition of template DNA on to the solid array support. The template DNA can even be in the form of unpurified PCR product, one of the major advantages of this technique. Protein microarray First spotting DNA template

14. Part 3, Step 2: ActionAudio Narration 1 5 3 2 4 Description of the action As shown in the animatio n. The second spotting step involves the addition of the cell-free lysate directly on top of the first spot. Transcription and translation can begin only after the second spotting step. Protein microarray Second spotting DNA template Cell-free lysate First show the parallelogram with dots. Then show the hand moving down into one of the spots followed by appearance of text ‘second spotting’ and then disappearance of hand and text. That spot must then be zoomed into and the figure on top must be shown.

15. Part 3, Step 3: ActionAudio Narration 1 5 3 2 4 Description of the action As shown in the animatio n. First show the grey surface with the green strands and the grey cloud on top. Then show the pink circles along with their label. These circles must move up as shown in the animation followed by appearance of the arrow. The pale purple ‘protein’ must then appear followed by the figure labelled as ‘tagged antibody’. This figure must move diagonally as shown until it binds to the pink shape. The protein expressed from the template DNA binds to the array surface by means of non- specific interactions, one of the drawbacks of this procedure. A detection antibody specific to the protein of interest is then added which indicates protein expression levels by means of a suitable fluorophore. Cell-free lysate Expressed protein Tagged detection antibody mRNA Ribosomes RNA Polymerase

16. Master Layout (Part 4) 5 3 2 4 1 This animation consists of 5 parts: Part 1 – Protein in situ array (PISA) Part 2 – Nucleic Acid Programmabe Protein Array (NAPPA) Part 3 – Multiple Spotting Technique (MIST) Part 4 – DNA Array to Protein Array (DAPA) Part 5 – HaloTag technique Ni-NTA coated slide DNA template Lysate- containing permeable membrane Tagged, expressed protein Protein tag-capturing agent He, M., Stoevesandt, O., Palmer, E. A., Khan, F. et al., Printing protein arrays from DNA arrays. Nat. Methods 2008, 5, 175–177.

17. Definitions of the components: Part 4 – DNA array to protein array (DAPA) 5 3 2 4 1 1. Ni-NTA coated slide: The microarray slide surface is coated with Nickel-nitrilotriacetic acid (Ni-NTA) which acts as a useful capture agent. 2. DNA template: PCR amplified DNA that codes for the protein of interest is immobilized on a Ni-NTA coated slide. This slide can be reused several times for generation of protein. 3. Lysate containing permeable membrane: A permeable membrane that is soaked with the cell-free extract is placed in between the immobilized DNA template slide and a slide having the protein tag- capturing agent. The newly expressed proteins penetrate the membrane and bind to the protein purification slide. 4. Tagged, expressed protein: The newly expressed protein bearing the tag molecule that is formed in the permeable membrane, slowly penetrates the membrane and gets immobilized by means of its tag- capturing agent. 5. Protein tag-capturing agent: A molecule that will specifically bind to the protein of interest thereby immobilizing it onto the Ni-NTA coated slide.

18. Part 4, Step 1: ActionAudio Narration 1 5 3 2 4 Description of the action As shown in the animatio n. First show the slide on top and the slide below along with the grey oval in between. Then show the dark green circles on the red strands which must move downwards as shown. Then show appearance of the maroon strands and then the green figures on top of it. This must move from left to right as shown. Then show appearance of the light blue figure (protein) which must slowly move down until it gets captured by the dark blue pie-shaped circles below. The slides bearing the DNA template and the protein tag-capturing agent are assembled face-to-face with a lysate containing permeable membrane placed in between. The expressed protein slowly penetrates the membrane and gets immobilized on the slide surface through its capture agent. The DNA template array can be reused several times in this method. Ni-NTA coated slide DNA template Lysate- containing permeable membrane Tagged, expressed protein Protein tag-capturing agent

19. Master Layout (Part 5) 5 3 2 4 1 This animation consists of 5 parts: Part 1 – Protein in situ array (PISA) Part 2 – Nucleic Acid Programmabe Protein Array (NAPPA) Part 3 – Multiple Spotting Technique (MIST) Part 4 – DNA Array to Protein Array (DAPA) Part 5 – HaloTag technique Transcription Translation DNA construct Ribosomes mRNA HaloTag bound protein HaloTag ligand Cell-free lysate Nath, N., Hurst, R., Hook, B., Meisenheimer, P. et al., Improving protein array performance: Focus on washing and storage conditions. J. Proteome Res. 2008, 7, 4475–4482.

20. Definitions of the components: Part 5 – HaloTag technique 5 3 2 4 1 1. DNA construct: The template DNA coding for the protein of interest along with the HaloTag. 2. HaloTag bound protein: HaloTag is a 33kD engineered derivative of bacterial hydrolase that can be used to tag the desired protein. The mode of interaction between HaloTag and its ligand is through covalent bonding, thereby ensuring firm capture of the protein on the array surface without any material loss during washing. It also prevents any loss of protein function as it allows for oriented capture of the protein on to the array surface. 3. HaloTag ligand: The PEG-coated glass slide is activated with the HaloTag ligand for oriented and firm capture of the expressed protein on to the array surface.

21. Part 5, Step 1: ActionAudio Narration 1 5 3 2 4 Description of the action Protein microarray Array surface One of the spots on the protein microarray must be zoomed into and the rest must be shown. First show the parallelogram with dots as shown. Zoom into one of the dots and show the figure drawn above. The slide is activated with the HaloTag ligand which captures the expressed protein through firm covalent interactions thereby preventing any material loss and ensuring oriented capture of the protein. HaloTag ligand

22. Part 5, Step 2: ActionAudio Narration 1 5 3 2 4 Description of the action Transcription Translation Ribosomes mRNA RNA Polymerase (part of cell-free lysate) As shown in the animation First show the purple strands followed by the green oval moving along them and then the appearance of the blue strands. Next show the pink round structures moving along the blue strands and appearance of the yellow protein shapes. The green shape on the yellow protein must then move down and get attached to the blue tags at the bottom as shown. The HaloTag fused protein is expressed using lysates like RRL or WGE and covalently captured on to the array surface through the HaloTag ligand. The specific interaction ensures oriented capture of the protein thereby preventing any possible functional loss. HaloTag ligand HaloTag bound protein DNA construct Firm covalent capture

23. Interactivity option 1:Step No:1 Boundary/limitsInteracativity Type Options Results 1 2 5 3 4 Arrange the following components of cell-free expression in the right order for protein synthesis to occur. A) Transcription initiation factors B) Translation factors E) Template DNA encoding tag sequence F) Cell-free lysate containing enzymes & ribosomes D)Amino acids C) Tagged array surface 1 2 3 4 5 6 Drag and drop User must be allowed to drag and drop the images given above into the numbered circles below. The correct order is E, F, A, B, D, C. If the user gets this right, there must be a pop-up which says ‘Correct answer’ else a popup saying ‘incorrect, try again’. User can try until he gets the correct answer.

24. Questionnaire 1 5 2 4 3 1. Which of the following techniques allows the DNA template slide to be reused several times? Answers: a) NAPPA b) MIST c) DAPA d)‏ HaloTag 2. Which technique ensures oriented capture of the expressed protein? Answers: a) PISA b) HaloTag c) NAPPA d)‏ MIST 3. Traditional cell-based techniques for microarray generation suffer from which of the following drawbacks? Answers: a) Protein expression in heterologous systems b) Protein purification c) Maintaining protein stability d)‏ All of the above 4. What is one of the major drawbacks of the NAPPA technique? Answers: a) It cannot generate high density arrays b) The immobilized DNA arrays cannot be stored for long c) It requires large volumes of cell-free lysate d)‏ Cloning procedures need to be carried out to generate cDNA. 5. Which of the following techniques can make use of unpurified PCR generated DNA product? Answers: a) PISA b) NAPPA c) DAPA d)‏ MIST

25. Links for further reading Books: 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. He, M., Stoevesandt, O., Taussig, M. J., In situ synthesis of protein arrays. Curr. Opin. Biotechnol. 2008, 19, 4–9. Jackson, A. M., Boutell, J., Cooley, N., He, M., Review: cell-free protein synthesis for proteomics. Brief Funct. Genom.Proteomic 2004, 2, 308–319. He, M., Taussig, M. J., Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method). Nucleic Acids Res. 2001, 29, e73. Ramachandran, N., Hainsworth, E., Bhullar, B., Eisenstein,S. et al., Self-assembling protein mircoarrays. Science 2004,305, 86–90.

26. Links for further reading Research papers: 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. Angenendt, P., Kreutzberger, J., Glokler, J., Hoheisel, J. D., Generation of high density protein microarrays by cell-free in situ expression of unpurified PCR products. Mol. Cell.Proteomics 2006, 5, 1658–1666. He, M., Stoevesandt, O., Palmer, E. A., Khan, F. et al., Printing protein arrays from DNA arrays. Nat. Methods 2008, 5, 175–177. Nath, N., Hurst, R., Hook, B., Meisenheimer, P. et al., Improving protein array performance: Focus on washing and storage conditions. J. Proteome Res. 2008, 7, 4475–4482.