On-chip protein synthesis for making microarrays.
Protein microarrays are a miniaturized format for displaying in close spatial density hundreds or thousands of purified proteins that provide a powerful platform for the high-throughput assay of protein function. The traditional method of producing them requires the high-throughput production and printing of proteins, a laborious method that raises concerns about the stability of the proteins and the shelf life of the arrays. A novel method of producing protein microarrays, called nucleic acid programmable protein array (NAPPA), overcomes these limitations by synthesizing proteins in situ. NAPPA entails spotting plasmid DNA encoding the relevant proteins, which are then simultaneously transcribed and translated by a cell-free system. The expressed proteins are captured and oriented at the site of expression by a capture reagent that targets a fusion protein on either the N- or C-terminus of the protein. Using a mammalian extract, NAPPA expresses and captures 1000-fold more protein per feature than conventional protein-printing arrays. Moreover, this approach minimizes concerns about protein stability and integrity, because proteins are produced just in time for assaying. NAPPA has already proven to be a robust tool for protein functional assays.
Ramachandran
Niroshan
N
Department of Biological Chemistry and Molecular Pharmacology, Harvard Institute of Proteomics, Harvard Medical School, Cambridge, MA, USA.
Hainsworth
Eugenie
E
Demirkan
Gokhan
G
LaBaer
Joshua
J
D000818:Animals; D019921:Biotinylation; D002474:Cell-Free System; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D010957:Plasmids / Q000378:metabolism; D040081:Protein Array Analysis / Q000295:instrumentation* / Q000379:methods*; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*