12/11/12 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 Proteomics Course NPTEL 1 12/11/12 IIT Bombay 3 Proteomics Course NPTEL IIT Bombay Proteomics Course NPTEL 2 12/11/12 • 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 Proteomics Course NPTEL He and Taussig. J Immunol Methods 2003, 274, 265 IIT Bombay 6 Proteomics Course NPTEL 3 12/11/12 • 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 Proteomics Course NPTEL Tag-capturing agent Array surface Protein microarray IIT Bombay 8 Proteomics Course NPTEL 4 12/11/12 mRNA Transcription Ribosomes Translation PCR generated DNA construct Tagged protein RNA Polymerase (part of cell-free lysate) Tag-capturing agent IIT Bombay 9 Proteomics Course NPTEL Transcription Translation His6 Tag Ni-NTA PCR DNA encoding N- or C-terminal tag sequence expressed; bound protein gets specifically captured by tag-capturing agent IIT Bombay 10 Proteomics Course NPTEL 5 12/11/12 IIT Bombay 11 Proteomics Course NPTEL • 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 Proteomics Course NPTEL 6 12/11/12 Ramachandran et al. Science 2004, 305, 86 Ramachandran et al. Nat Methods 2008, 5, 535 IIT Bombay 13 Proteomics Course NPTEL • 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 Proteomics Course NPTEL 7 12/11/12 GS GS GS T cDNA + T T GST tag Anti-GST BSA antibody BS3 Aminosilane coated glass slide NAPPA master mix Protein microarray IIT Bombay 15 Proteomics Course NPTEL Ribosomes mRNA Translation Transcription GST tag RNA Polymerase (part of cell-free cDNA + GST GST lysate) GST tag GST Anti-GST antibodies BSA BS3 IIT Bombay 16 Proteomics Course NPTEL 8 12/11/12 Capture antibody (e.g. anti-GST) GST GST Expressed cDNA protein (& GST tag) Cell free expression BSA of target protein BS3 IIT Bombay 17 Proteomics Course NPTEL Anti-GST Anti-p21 Cdk2 CDT1 Fos Cdk4 Cdk6 Jun p21 CycD1 p21 IIT Bombay 18 Proteomics Course NPTEL 9 12/11/12 Pri. Sec. IVTT mix Block Ab Ab TSA ο 30 C/90 min RT/ 45 min RT/ 45 min RT/ 10 min 15οC/30 min Blocking Expression Primary Secondary TSA Antibody Antibody Detection IIT Bombay 19 Proteomics Course NPTEL • 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 Proteomics Course NPTEL 10 12/11/12 Transcription factors Kinases Membrane proteins 96% (136/141) 97% (38/39) 93% (239/258) No protein class or size biases Very tight range of protein levels Size < 50 kDa Size 50-100 kDa Size < 100 kDa 98% (617/631) 92% (253/275) 88% (30/34) Ramachandran et al. Nat Methods 2008, 5, 535 IIT Bombay 21 Proteomics Course NPTEL • 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 Proteomics Course NPTEL 11 12/11/12 • 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 Proteomics Course NPTEL IIT Bombay 24 Proteomics Course NPTEL 12 12/11/12 Angenendt et al. Mol Cell Proteomics 2006, 5, 1658 IIT Bombay 25 Proteomics Course NPTEL • 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 Proteomics Course NPTEL 13 12/11/12 DNA template First spotting Protein microarray IIT Bombay 27 Proteomics Course NPTEL Cell-free lysate DNA template Second spotting Protein microarray IIT Bombay 28 Proteomics Course NPTEL 14 12/11/12 Ribosomes mRNA Tagged detection antibody Expressed protein Cell-free lysate RNA Polymerase IIT Bombay 29 Proteomics Course NPTEL 2nd spotting Detection 1st spotting step IIT Bombay 30 Proteomics Course NPTEL 15 12/11/12 IIT Bombay 31 Proteomics Course NPTEL • 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 Proteomics Course NPTEL 16 12/11/12 IIT Bombay 33 Proteomics Course NPTEL • 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 Proteomics Course NPTEL 17 12/11/12 • 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 Proteomics Course NPTEL Ni-NTA coated slide DNA template Permeable membrane Lysate- containing permeable Tagged, membrane expressed protein Protein tag-capturing agent Ni-NTA coated slide IIT Bombay 36 Proteomics Course NPTEL 18 12/11/12 IIT Bombay 37 Proteomics Course NPTEL • 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 Proteomics Course NPTEL 19 12/11/12 Nath et al., J. Proteome Res. 2008, 7, 4475 IIT Bombay 39 Proteomics Course NPTEL • 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 PEG- coated slide, activated with HaloTag ligand • Enables oriented capture of proteins • ensuring no loss of function IIT Bombay 40 Proteomics Course NPTEL 20 12/11/12 HaloTag ligand Array surface Protein microarray IIT Bombay 41 Proteomics Course NPTEL mRNA Transcription Ribosomes Translation DNA construct HaloTag bound RNA Polymerase protein (part of cell-free lysate) Firm HaloTag covalent ligand capture IIT Bombay 42 Proteomics Course NPTEL 21 12/11/12 Halotag ligand IIT Bombay 43 Proteomics Course NPTEL IIT Bombay 44 Proteomics Course NPTEL 22 12/11/12 • 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 Proteomics Course NPTEL Cell-free expression PISA microarray NAPPA MIST HaloTag Arrays DAPA Figure 1 23 12/11/12 • 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 Proteomics Course NPTEL • 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. • 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. IIT Bombay 48 Proteomics Course NPTEL 24 12/11/12 • 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. • Katzen, F., Chang, G., Kudlicki, W. The past, present and future of cell-free protein synthesis. Trends Biotechnol. 2005, 23 (3), 150-156. • 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 84-88.