Cell-free protein array: Difference between revisions

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Revision as of 19:53, 10 August 2009

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'''Cell-free protein array''' technology produces [[protein microarray]]s by performing [[in vitro]] synthesis of the target proteins from their [[DNA]] templates. This method of synthesizing protein microarrays overcomes the many obstacles and challenges faced by traditional methods of protein array production<ref name="Stevens, R. C. 2000">Stevens, R. C. (2000). "Design of high-throughput methods of protein production for structural biology." Structure 8(9): R177-R185.</ref> that have prevented widespread adoption of protein microarrays in [[proteomics]]. Protein arrays made from this technology can be used for testing [[protein-protein interactions]], as well as protein interactions with other cellular molecules such as DNA and lipids. Other applications include enzymatic inhibition assays and screenings of antibody specificity.

==Overview / background==

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Various research groups have developed their own methods, each differing in their approach, but can be summarized into 3 main groups.

[[Image:Figure_1_NAPPAFigure 1 NAPPA.png|thumb|600px|'''Figure 1: Schematic diagram of NAPPA''']]

;Nucleic acid programmable protein array (NAPPA): NAPPA<ref>Ramachandran, N., E. Hainsworth, et al. (2004). "Self-assembling protein microarrays." Science 305(5680): 86-90.</ref> uses DNA template that has already been immobilized onto the same protein capture surface. The DNA template is [[biotinylation|biotinylated]] and is bound to [[avidin]] that is pre-coated onto the protein capture surface. Newly synthesized proteins which are tagged with GST are then immobilized next to the template DNA by binding to the adjacent polyclonal anti-GST capture antibody that is also pre-coated onto the capture surface (Figure 1). The main drawback of this method is the extra and tedious preparation steps at the beginning of the process: (1) the [[molecular cloning|cloning]] of [[CDNA|cDNAscDNA]]s in an expression-ready [[expression vector|vector]]; and (2) the need to biotinylate the [[plasmid]] DNA but not to interfere with transcription. Moreover, the resulting protein array is not ‘pure’ because the proteins are co-localized with their DNA templates and capture antibodies<ref>He, M., O. Stoevesandt, et al. (2008). "In situ synthesis of protein arrays." Curr Opin Biotechnol 19(1): 4-9.</ref>.

[[Image:Figure_2_PISAFigure 2 PISA.png|thumb|600px|'''Figure 2: Schematic diagram of PISA''']]

; Protein ''in situ'' array (PISA): Unlike NAPPA, PISA<ref>He, M. and M. J. Taussig (2001). "Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method)." Nucleic Acids Res 29(15): E73-3.</ref> completely bypasses DNA immobilization as the DNA template is added as a free molecule in the reaction mixture. In 2006, another group refined and miniaturized this method by using multiple spotting technique to spot the DNA template and cell-free transcription and translation mixture on a high-density protein microarray with up to 13,000 spots<ref>Angenendt, P., J. Kreutzberger, et al. (2006). "Generation of high density protein microarrays by cell-free in situ expression of unpurified PCR products." Mol Cell Proteomics 5(9): 1658-66.</ref> (Figure 2). This was made possible by the automated system used to accurately and sequentially supply the reagents for the transcription/translation reaction occurs in a small, sub-nanolitre droplet.

[[Image:Figure_3_puromycin2Figure 3 puromycin2.png|thumb|600px|'''Figure 3: Schematic diagram of ''In situ'' puromycin-capture''']]

; ''In situ'' puromycin-capture: This method is an adaptation of [[mRNA display]] technology. [[PCR]] DNA is first transcribed to [[mRNA]], and a single-stranded DNA [[oligonucleotide]] modified with [[biotin]] and [[puromycin]] on each end is then hybridized to the 3’-end of the mRNA. The mRNAs are then arrayed on a slide and immobilized by the binding of biotin to [[streptavidin]] that is pre-coated on the slide. Cell extract is then dispensed on the slide for ''in situ'' translation to take place. When the ribosome reaches the hybridized oligonucleotide, it stalls and incorporates the puromycin molecule to the nascent [[polypeptide]] chain, thereby attaching the newly synthesized protein to the microarray via the DNA oligonucleotide<ref>Tao, S. C. and H. Zhu (2006). "Protein chip fabrication by capture of nascent polypeptides." Nat Biotechnol 24(10): 1253-4.</ref> (Figure 3). A pure protein array is obtained after the mRNA is digested with [[RNase]]. The protein spots generated by this method are very sharply defined and can be produced at a high density.

===Nano-well array format===

[[Image:Figure_4_nano_wellFigure 4 nano well.png|thumb|600px|'''Figure 4: Schematic diagram of the nano-well array format''']]

Nano-well array formats are used to express individual proteins in small volume reaction vessels or nano-wells<ref>Angenendt, P., L. Nyarsik, et al. (2004). "Cell-free protein expression and functional assay in nanowell chip format." Anal Chem 76(7): 1844-9.</ref><ref>Kinpara, T., R. Mizuno, et al. (2004). "A picoliter chamber array for cell-free protein synthesis." J Biochem 136(2): 149-54.</ref> (Figure 4). This format is sometimes preferred because it avoids the need to immobilize the target protein which might result in the potential loss of protein activity. The miniaturization of the array also conserves solution and precious compounds that might be used in screening assays. Moreover, the structural properties of individual wells help to prevent cross-contamination among chambers.

=== DNA array to protein array (DAPA) ===

[[Image:Figure_5_DAPAFigure 5 DAPA.png|thumb|600px|'''Figure 5: Schematic diagram of DAPA''']]

DNA array to protein array (DAPA) is a method developed in 2007 to repeatedly produce protein arrays by ‘printing’ them from a single DNA template array, on demand<ref>He, M., O. Stoevesandt, et al. (2008). "Printing protein arrays from DNA arrays." Nat Methods 5(2): 175-7.</ref> (Figure 5). It starts with the spotting and immobilization of an array of DNA templates onto a glass slide. The slide is then assembled face-to-face with a second slide pre-coated with a protein-capturing reagent, and a membrane soaked with cell extract is placed between the two slides for transcription and translation to take place. The newly-synthesized his-tagged proteins are then immobilized onto the slide to form the array. Over 20 protein arrays can be printed from a single DNA array with no adverse effects on production efficiency.

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;Improves protein availability:

* Many proteins, including antibodies, are difficult to express in host cells due to problems with insolubility, [[disulfide]] bonds or host cell toxicity<ref> name="Stevens, R. C. (2000). "Design of high-throughput methods of protein production for structural biology." Structure 8(9): R177-R185.</ref>. Cell-free protein array makes many of such proteins available for use in protein microarrays.

;Enables long term storage

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==Limitation==

*'''[[Post-translational modification]]''' of proteins in proteins generated by cell-free protein synthesis <ref>Promega ''in vitro'' Expression Guide [http://www.promega.com/guides/ive_guide/ivex_chp8.pdf Promega ''in vitro'' Expression Guide]</ref> is still limited compared to the traditional methods<ref>Chatterjee, D.K. and J. LaBaer (2006). "Protein technologies." Curr Opin Biotech 17(4): 334-336.</ref>, and may not be as biologically relevant.

==Applications==

*'''Protein interactions''': To screen for [[protein-protein interactions]]<ref>Ramachandran, N., E. Hainsworth, et al. (2004). "Self-assembling protein microarrays." Science 305(5680): 86-90.</ref> and protein interactions with other molecules such as [[metabolite]]s, [[lipid]]s, DNA and small molecules<ref>He, M. and M. W. Wang (2007). "Arraying proteins by cell-free synthesis." Biomol Eng 24(4): 375-80.</ref>.

*'''Enzyme inhibition assay'''<ref>Angenendt, P., L. Nyarsik, et al. (2004). "Cell-free protein expression and functional assay in nanowell chip format." Anal Chem 76(7): 1844-9.</ref>: For high throughput drug candidate screening and to discover novel [[enzyme]]s for use in [[biotechnology]].

*'''Screening antibody specificity'''<ref>He, M. and M. J. Taussig (2003). "DiscernArray technology: a cell-free method for the generation of protein arrays from PCR DNA." J Immunol Methods 274(1-2): 265-70.</ref>