PMC:4996395 / 4543-8021
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4996395","sourcedb":"PMC","sourceid":"4996395","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4996395","text":"2. Concept of Protein Microarrays\nIn 2004, LaBaer’s lab designed and developed a novel protein microarray, termed NAPPA, based on shuttling cDNA clones into expression plasmids—typically using Gateway technology—adding a transcriptional promoter and also an in-frame polypeptide capture tag.\nCloning the cDNAs into a specialized vector requires a much greater upfront investment compared to the conventional Polymerase Chain Reaction (PCR). However, there are several advantages over typical protein microarrays that establish NAPPA technology as a powerful platform: (i) the production of a glycerol stock for the clone allows the maintenance of the gene integrity indefinitely; (ii) it ensures high fidelity since the clone sequence is verified; and (iii) inserting the clones into plasmids permits the incorporation of tags and antibiotic resistance genes for specific selection. Generally, proteins are fused with glutathione-S-transferase (GST) in NAPPA technology; however, other tags such as flag, hemagglutinin (HA), c-Myc, and Halo tags have been used for specific applications.\nBacteria cultures are employed as hosts for the high quality supercoiled plasmid DNA of interest. Thus, after the purification of the DNA plasmids, these are printed onto an activated ester surface along with a homo-bifunctional crosslinker (BS3, SMCC…), bovine serum albumin (BSA) and anti-tag antibody. BSA efficiently increases the DNA binding and reduces the unspecific interactions, whereas the anti-tag antibody attaches the expressed protein [10]. When the cell-free expression system is added to the array, a coupled transcription/translation reaction results and the nascent protein is linked to the capture agent tag through the C-terminal end assuring the complete translation of the protein (Figure 2). A cell-free expression system produces a protein by using biomolecular translation machinery without the usage of living cells. The reaction solution includes the transcriptional and translational molecular machinery consisting of RNA polymerases for mRNA transcription, ribosomes for polypeptide translation, tRNA and amino acids, enzymatic cofactors, an energy source, and cellular components essential for proper protein folding.\nFigure 2 Scanning images showing the spots corresponding to DNA printed onto the surface before the protein expression (A) and the spots for the expressed proteins after the incubation with the anti-tag antibody (B). In an updated version of NAPPA, LaBaer and colleagues built an array of 1000 human genes and demonstrated that the vast majority of these genes (~96%) showed a detectable protein signal. In addition, they concluded that this platform is unbiased in relation to protein size—signal intensities were independent from molecular size—enabling unbiased study of protein function in a HT manner. In turn, they demonstrated their high stability since the DNA is more stable than proteins. Moreover, there is high intra- and inter-protein display reproducibility in these kinds of arrays [9]. It is also remarkable that NAPPA is the only in situ protein technology that has been widely employed in biological and biomedical research studies. To date, more than 30,000 different proteins have been produced on NAPPA arrays, including whole proteomes of several microorganisms and \u003e12,000 different full-length human proteins. All in all, thousands of NAPPAs can be produced per year thanks to the automation developed in the field.","divisions":[{"label":"Title","span":{"begin":0,"end":33}},{"label":"Figure caption","span":{"begin":2236,"end":2455}}],"tracks":[{"project":"2_test","denotations":[{"id":"27600221-15232106-69478167","span":{"begin":1538,"end":1540},"obj":"15232106"},{"id":"27600221-18469824-69478168","span":{"begin":3037,"end":3038},"obj":"18469824"}],"attributes":[{"subj":"27600221-15232106-69478167","pred":"source","obj":"2_test"},{"subj":"27600221-18469824-69478168","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#cd93ec","default":true}]}]}}