Cell-free expression based microarrays

Transcription

1 Dr. Sanjeeva Srivastava IIT Bombay Cell-free expression based microarrays Protein in situ array (PISA) Nucleic Acid Programmable Protein Array (NAPPA) Multiple Spotting Technique (MIST) DNA Array to Protein Array (DAPA) HaloTag Array IIT Bombay 2 1

2 IIT Bombay 3 IIT Bombay 2

3 Able to utilize wide variety of DNA templates PCR products or plasmids Process should be simple, quick and cost-effective Avoid storage effects Simultaneous production of thousands of proteins in single reaction Methods to detect & analyze bound protein simple IIT Bombay 5 He and Taussig. J Immunol Methods 2003, 274, 265 IIT Bombay 6 3

4 DNA construct produced by PCR T7 promoter, sequences for translation initiation (Shine-Dalgarno or Kozak), an N- or C-terminal tag for immobilization, suitable termination sequences Used hexahistidine (His6) binding sequences and microtiter plate coated with Ni-NTA Protein expression with E. coli S30 or RRL After translation, protein bound specifically on surface through tag sequence IIT Bombay 7 Tag-capturing agent Array surface Protein microarray IIT Bombay 8 4

5 Transcription mrna Ribosomes PCR generated DNA construct RNA Polymerase (part of cell-free lysate) Translation Tagged protein Tag-capturing agent IIT Bombay 9 Transcription Translation Ni-NTA His 6 Tag PCR DNA encoding N- or C-terminal tag sequence expressed; bound protein gets specifically captured by tag-capturing agent IIT Bombay 10 5

6 IIT Bombay 11 Merits Protein purification not required Rapid, single step process Specific protein attachment Soluble proteins formed Demerits Possible loss of function during immobilization Relatively high volume of cell-free lysate required IIT Bombay 12 6

7 Ramachandran et al. Science 2004, 305, 86 Ramachandran et al. Nat Methods 2008, 5, 535 IIT Bombay 13 Plasmid encoding target proteins fused with an affinity tag are affixed to surface Array is activated by the addition of a cell free expression system Target proteins are expressed and immobilized in situ, and detected using a universal anti-tag antibody IIT Bombay 14 7

8 BSA cdna + GST tag GS T GS T GS T Anti-GST antibody Aminosilane coated glass slide BS 3 NAPPA master mix Protein microarray IIT Bombay 15 Ribosomes mrna Transcription Translation GST tag cdna + GST tag GST GST GST RNA Polymerase (part of cell-free lysate) Anti-GST antibodies BSA BS 3 IIT Bombay 16 8

9 cdna (& GST tag) Capture antibody (e.g. anti-gst) GST Cell free expression GST Expressed protein BSA BS3 of target protein IIT Bombay 17 Anti-GST Anti-p21 Cdk2 CDT1 Fos Cdk4 Cdk6 Jun p21 CycD1 p21 IIT Bombay 18 9

10 IVTT mix Block Pri. Ab Sec. Ab TSA 30 ο C/90 min 15 ο C/30 min RT/ 45 min RT/ 45 min RT/ 10 min Blocking Expression Primary Antibody Secondary Antibody TSA Detection IIT Bombay 19 In high-density NAPPA arrays > 95% of proteins express and capture well > 10,000 proteins tested Multiple organisms tested Membrane proteins express well IIT Bombay 20 10

11 Transcription factors 96% (136/141) Kinases 97% (38/39) Membrane proteins 93% (239/258) No protein class or size biases Very tight range of protein levels Size < 50 kda 98% (617/631) Size kda 92% (253/275) Size < 100 kda 88% (30/34) Ramachandran et al. Nat Methods 2008, 5, 535 IIT Bombay 21 Merits No need to express and purify protein separately Expression in mammalian milieu (natural folding) Proteins produced just-in-time for assay Shelf life not an issue Access to all cloned cdnas Express & capture more than protein spotting arrays Retains functionality of traditional protein arrays Arrays stable on bench until activated IIT Bombay 22 11

12 Demerits Cloning procedure required Pure protein array not produced Peptide tags may lead to sterical effects blocking important binding domains Functionality of proteins? IIT Bombay 23 IIT Bombay 24 12

13 Angenendt et al. Mol Cell Proteomics 2006, 5, 1658 IIT Bombay 25 1st spotting step - addition of DNA template onto solid support 2nd spotting step - cell-free expression mixture transferred directly on top of first spot Proteins immobilized on activated array surface after translation by means of a tag-capturing agent or non-specific ionic interactions IIT Bombay 26 13

14 DNA template First spotting Protein microarray IIT Bombay 27 Cell-free lysate DNA template Second spotting Protein microarray IIT Bombay 28 14

15 Ribosomes mrna Tagged detection antibody Expressed protein RNA Polymerase Cell-free lysate IIT Bombay 29 2 nd spotting Detection 1 st spotting step IIT Bombay 30 15

16 IIT Bombay 31 Merits Unpurified DNA products used as template Very high density protein arrays generated Demerits Loss of signal intensity with prolonged incubation time Non-specific protein binding Time consuming process IIT Bombay 32 16

17 IIT Bombay 33 PCR amplified DNA fragments encoding tagged protein immobilized onto a Ni-NTA coated slide and assembled face-to-face with another Ni- NTA slide bearing protein tag-capturing agent Repeated use of same DNA template slide to print multiple protein arrays IIT Bombay 34 17

18 Permeable membrane having cell-free lysate positioned in between the slides Protein synthesis took place from immobilized DNA spots Newly synthesized proteins penetrates membrane and bind to surface of capture slide IIT Bombay 35 Ni-NTA coated slide DNA template Permeable membrane Tagged, expressed protein Lysatecontaining permeable membrane Protein tag-capturing agent Ni-NTA coated slide IIT Bombay 36 18

19 IIT Bombay 37 Demerits Reusable DNA template array Pure protein array generated DNA template array can be stored for long durations Demerits Broadening of spots due to diffusion Not ascertained if multimeric proteins assemble effectively Time consuming process IIT Bombay 38 19

20 Nath et al., J. Proteome Res. 2008, 7, 4475 IIT Bombay 39 HaloTag - a 33 kda engineered derivative of bacterial hydrolase, used to tag desired protein Proteins fused with HaloTag expressed using WGE/RRL and covalently captured on a PEGcoated slide, activated with HaloTag ligand Enables oriented capture of proteins ensuring no loss of function IIT Bombay 40 20

21 HaloTag ligand Array surface Protein microarray IIT Bombay 41 Transcription mrna Ribosomes Translation DNA construct RNA Polymerase (part of cell-free lysate) HaloTag ligand HaloTag bound protein Firm covalent capture IIT Bombay 42 21

22 Halotag ligand IIT Bombay 43 IIT Bombay 44 22

23 Merits Strong covalent bond between protein and ligand No material loss during washing Oriented capture of protein No non-specific adsorption Easy quantification No need for a microarrayer printer Demerits Possible loss of function on binding to Halotag HT application will require optimization of printing IIT Bombay 45 PISA Cell-free expression microarray NAPPA HaloTag Arrays MIST DAPA Figure 1 23

24 Cell-free microarrays principle, merits and demerits Protein in situ array (PISA) Nucleic Acid Programmable Protein Array (NAPPA) Multiple Spotting Technique (MIST) DNA Array to Protein Array (DAPA) HaloTag Array IIT Bombay 47 Chandra, H. & Srivastava, S. Cell-free synthesis-based protein microarrays and their applications. Proteomics 2010, 10, 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, 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., Selfassembling protein mircoarrays. Science 2004,305, Ramachandran, N., Raphael, J. V., Hainsworth, E., Demirkan,G. et al., Nextgeneration high-density self-assembling functional protein arrays. Nat. Methods 2008, 5, IIT Bombay 48 24

25 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, He, M., Stoevesandt, O., Palmer, E. A., Khan, F. et al., Printing protein arrays from DNA arrays. Nat. Methods 2008, 5, 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, Katzen, F., Chang, G., Kudlicki, W. The past, present and future of cell-free protein synthesis. Trends Biotechnol. 2005, 23 (3), Amita Nand, Anju Gautam, Javier Batista Pérez, Alejandro Merino, Jinsong Zhu. Emerging technology of in situ cell free expression protein microarrays. Protein & Cell. February 2012, Volume 3, Issue 2, pp IIT Bombay 49 Dr. Joshua LaBaer and laboratory members at Harvard Institute of Proteomics for recombinational cloning and NAPPA microarray information. CSHL, New York, Proteomics course IIT Bombay 50 25