Cell-free protein array: Difference between revisions

Revision as of 18:54, 19 March 2008

Latest revision as of 00:20, 28 May 2025

(46 intermediate revisions by 27 users not shown)

Line 1:

{{Short description|DNA technology}}

'''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 microarray]]s overcomes the many obstacles and challenges faced by traditional methods of protein array production<ref>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.

'''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 microarray]]smicroarrays 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-proteinprotein–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==

==Overview /and background==

The runaway success of [[DNA microarraysmicroarray]]s has generated much enthusiasm for protein microarrays. However, protein microarrays have not quite taken off as expected, even with the necessary tools and know-how from DNA microarrays being in place and ready for adaptation. One major reason is that protein microarrays are much more laborious and technically challenging to construct than DNA microarrays.

The traditional methods of producing protein arrays require the separate [[in vivo|''in vivo'']] expression of hundreds or thousands of proteins, followed by separate purification and immobilization of the proteins on a solid surface. Cell-free protein array technology attempts to simplify protein microarray construction by bypassing the need to express the proteins in [[bacteria]] cells and the subsequent need to purify them. It takes advantage of available [[cell-free protein synthesis]] technology which has demonstrated that protein synthesis can occur without an intact cell as long as cell extracts containing the DNA template, [[transcription (genetics)|transcription]] and [[translation (biology)|translation]] raw materials and machinery are provided<ref>Katzen, F., G. Chang, et al. (2005). "The past, present and future of cell-free protein synthesis." Trends Biotechnol 23(3): 150-6.</ref>. Common sources of cell extracts used in cell-free protein array technology include [[wheat germ]], [[Escherichia coli|''Escherichia coli'']], and rabbit [[reticulocyte]]. Cell extracts from other sources such as [[hyperthermophiles]], [[hybridoma|hybridomas]], [[Xenopus]] [[oocyte|oocytes]], insect, mammalian and human cells have also been used<ref>He, M., O. Stoevesandt, et al. (2008). "In situ synthesis of protein arrays." Curr Opin Biotechnol 19(1): 4-9.</ref>.

The traditional methods of producing protein arrays require the separate ''[[in vivo|]]''in vivo'']] expression of hundreds or thousands of proteins, followed by separate purification and immobilization of the proteins on a solid surface. Cell-free protein array technology attempts to simplify protein microarray construction by bypassing the need to express the proteins in [[bacteria]] cells and the subsequent need to purify them. It takes advantage of available [[cell-free protein synthesis]] technology which has demonstrated that protein synthesis can occur without an intact cell as long as cell extracts containing the DNA template, [[transcription (genetics)|transcription]] and [[translation (biology)|translation]] raw materials and machinery are provided.<ref>Katzen, F., G. Chang, et al. (2005). "The past, present and future of cell-free protein synthesis." Trends Biotechnol 23(3): 150-6150–6.</ref>. Common sources of cell extracts used in cell-free protein array technology include [[wheat germ]], ''[[Escherichia coli|]]''Escherichia coli'']], and rabbit [[reticulocyte]]. Cell extracts from other sources such as [[hyperthermophileshyperthermophile]]s, [[hybridoma|hybridomas]]s, [[Xenopus]] [[oocyte|oocytes]]s, insect, mammalian and human cells have also been used.<ref name="O. Stoevesandt, 2008">He, M., O. Stoevesandt, et al. (2008). "In situ synthesis of protein arrays." Curr Opin Biotechnol 19(1): 4-94–9.</ref>.

The target proteins are synthesized [[in situ|''in situ'']] on the protein microarray, directly from the DNA template, thus skipping many of the steps in traditional protein microarray production and their accompanying technical limitations. More importantly, the expression of the proteins can be done in parallel, meaning all the proteins can be expressed together in a single reaction. This ability to multiplex protein expression is a major time-saver in the production process.

The target proteins are synthesized ''[[in situ|]]''in situ'']] on the protein microarray, directly from the DNA template, thus skipping many of the steps in traditional protein microarray production and their accompanying technical limitations. More importantly, the expression of the proteins can be done in parallel, meaning all the proteins can be expressed together in a single reaction. This ability to multiplex protein expression is a major time-saver in the production process.

==Methods of synthesis==

=== ''In situ'' methods ===

In the ''in situ'' method, protein synthesis is carried out on a protein array surface that is pre-coated with a protein-capturing reagent or [[antibody]]. Once the newly synthesized proteins are released from the [[ribosome]], the [[protein tag|tag sequence]] that is also synthesized at the [[N-terminus|N-]] or [[C-terminus]] of each nascent protein will be bound by the capture reagent or antibody, thus immobilizing the proteins to form an array. Commonly used tags include [[Polyhistidine-tag|polyhistidine]] (His)6 and [[Glutathione-S-transferase|glutathione s-transferase]] (GST).

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 diagramDiagram of NAPPA.''']]

====Nucleic acid programmable protein array (NAPPA)====

;NucleicNAPPA<ref acidname="Ramachandran, programmableN. protein array’ (NAPPA): NAPPA<ref2004">Ramachandran, N., E. Hainsworth, et al. (2004). "Self-assembling protein microarrays." Science 305(5680): 86-9086–90.</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 and,but atnot the same time, notto 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., name="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 diagramDiagram of PISA.''']]

====Protein ''in situ'' array (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): 1658-661658–66.</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 diagramDiagram of ''In situ'' puromycin-capture.''']]

====''In situ'' puromycin-capture====

; ''In situ'' puromycin-capture: This method is an adaptation of [[mRNA display]] technology. [[Polymerase chain reaction|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 hybrizedhybridized 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): 1253-41253–4.</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-wellNanowell array formats are used to express individual proteins in small volume reaction vessels or nano-wellsnanowells<ref name="Angenendt, P. 2004">Angenendt, P., L. Nyarsik, et al. (2004). "Cell-free protein expression and functional assay in nanowell chip format." Anal Chem 76(7): 1844-91844–9.</ref><ref>Kinpara, T., R. Mizuno, et al. (2004). "A picoliter chamber array for cell-free protein synthesis." J Biochem 136(2): 149-54149–54.</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. In 2012 an improved NAPPA was published, which used a nanowell array to prevent diffusion. Here the DNA was immobilized in the well together with an anti-GST antibody. Then cell-free expression mix was added and the wells closed by a lid. The nascent proteins containing a GST-tag were bound to the well surface enabling a NAPPA-array with higher density and nearly no cross-contaminations.<ref name="Takulapalli BR 2012">Takulapalli BR, Qiu J, et al. (2012). "High density diffusion-free nanowell arrays." J Proteome Res. 11(8):4382-91</ref>

[[Image:Figure_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): 175-7175–7.</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. OverIn the publication in 18 of 20 replications a protein arraysmicroarray copy could be generated. Potentially the process can be printedrepeated fromas aoften singleas needed, as long as the DNA arrayis withunharmed noby adverseDNAses, effectsdegradation onor productionmechanical efficiencyabrasion.

[[Image:Figure_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.

==Advantages==

Many of the advantages of cell-free protein array technology address the limitations of cell-based expression system used in traditional methods of protein microarray production.

;===Rapid and cost-effective:===

*AvoidsThe method avoids DNA cloning (with the exception of NAPPA) and can quickly convert genetic information into functional proteins by using PCR DNA. The reduced steps in production and the ability to miniaturize the system saves on reagent consumption and cuts production costs.

*The reduced steps in production and the ability to miniaturize the system saves on reagent consumption and cuts production costs.

;===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===

*Unlike DNA, which is a highly stable molecule, proteins are a heterogeneous class of molecules with different stability and physiochemical properties. Maintaining the proteins’ folding and function in an immobilized state over long periods of storage is a major challenge for protein microarrays. Cell-free methods provide the option to quickly obtaining protein microarrays on demand, thus eliminating any problems associated with long-term storage.

;===Flexible===

*AmenableThe method is amenable to a range of different templates: PCR products, plasmids and mRNA. Additional components can be included during synthesis to adjust the environment for protein folding, disulfide bond formation, modification or protein activity.<ref name="O. Stoevesandt, 2008"/>

*Additional components can be included during synthesis to adjust the environment for protein folding, disulfide bond formation, modification or protein activity<ref>He, M., O. Stoevesandt, et al. (2008). "In situ synthesis of protein arrays." Curr Opin Biotechnol 19(1): 4-9.</ref>.

==Limitation==

*'''[[Post-translational modification]]''' of proteins in proteins generated by cell-free protein synthesis <ref>[http://www.promega.com/guides/ive_guide/ivex_chp8.pdf Promega ''in vitro'' Expression Guide] [{{webarchive |url=https://web.archive.org/web/20071107082528/http://www.promega.com/guides/ive_guide/ivex_chp8.pdf] |date=November 7, 2007 }}</ref> is still limited compared to the traditional methods,<ref>Chatterjee, D.K. and J. LaBaer (2006). "Protein technologies." Curr Opin Biotech 17(4): 334-336334–336.</ref>, and may not be as biologically relevant.

==Applications==

Protein interactions: To screen for [[protein–protein interactions]]<ref name="Ramachandran, N. 2004"/> 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 name="Angenendt, P. 2004"/> 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>

*'''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 [[metabolites]], [[lipids]], 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 [[enzymes]] 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>

==References==

<ref name="Protein arrays: recent achievements and their application to study the human proteome.">{{cite journal | url=http://eurekaselect.com/113845/article | title=Welcome to Bentham Science Publisher | journal=Current Proteomics | volume=10 | issue=2 | pages=83–97 | last1=Casado-Vela | first1=Juan | last2=Gonzalez-Gonzalez | first2=Maria | last3=Matarraz | first3=Sergio | last4=Martinez-Esteso | first4=Maria Jose | last5=Vilella | first5=Maite | last6=Sayagues | first6=Jose Maria | last7=Fuentes | first7=Manuel | last8=Lacal | first8=Juan Carlos | doi=10.2174/1570164611310020003 | url-access=subscription }}</ref>

{{reflist}}

==External links==

*[https://web.archive.org/web/20090928065617/http://www.biodesign.asu.edu/labs/labaer/research NAPPA]

*[https://web.archive.org/web/20090105152214/http://www.discerna.co.uk/discerna_discerna_technologies_arrays.htm PISA and DAPA]

*[https://web.archive.org/web/20080220114252/http://www.functionalgenomics.org.uk/sections/resources/protein_arrays.htm Protein arrays resource page]

{{DEFAULTSORT:Cell-Free Protein Array}}

*[http://www.hip.harvard.edu/research/protein_microarray/index.htm NAPPA]

[[Category:Molecular biology]]

*[http://www.discerna.co.uk/discerna_discerna_technologies_arrays.htm PISA and DAPA]

[[Category:Microarrays]]

*[http://www.functionalgenomics.org.uk/sections/resources/protein_arrays.htm Protein arrays resource page]