12/11/12

Dr. Sanjeeva Srivastava IIT Bombay

• Cell-free expression based microarrays • 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 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 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

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 • 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

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

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

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 • 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 -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.

IIT Bombay 49 Proteomics Course NPTEL

• Dr. Joshua LaBaer and laboratory members at Harvard Institute of Proteomics for recombinational cloning and NAPPA microarray information. • CSHL, New York, Proteomics course 2008.

IIT Bombay 50 Proteomics Course NPTEL

25