Photocleavage-based affinity purification and printing of cell-free expressed proteins: application to proteome microarrays.
Proteome microarrays hold great promise for various biotechnological and biomedical applications, including mapping protein-protein interactions, drug discovery, and biomarker discovery. However, the need to express, purify, and print thousands of functional proteins at high density on a microarray substrate presents challenges that limit their widespread availability and use. We report the development of new methods, based on photocleavage, for the purification and printing of nascent proteins. Photocleavable biotin (PC-biotin) is incorporated into nascent proteins by misaminoacylated transfer RNAs (tRNAs) used in a coupled transcription/translation rabbit reticulocyte cell-free expression system. Proteins were affinity isolated onto (strept)avidin-coated beads and then photoreleased (PC-SNAG). Compared with polyhistidine tag-based affinity purification, PC-SNAG provided a higher purity yet a comparable yield using a glutathione-S-transferase (GST) test protein. Antibody-mediated PC-SNAG is also demonstrated. PC-SNAG proteins were found to exhibit native enzymatic activity and were suitable for the printing of ordered protein microarrays used in protein-protein interaction assays. Alternatively, when beads carrying photocleavably tethered proteins were placed in close proximity to an activated planar surface and then illuminated, proteins were transferred directly to the surface (PC-PRINT) to form discrete spots whose dimensions match those of the beads. PC-PRINT can provide an inexpensive method to fabricate very large-scale, high-density proteome microarrays. Moreover, transferring the proteins off the beads significantly reduces background autofluorescence observed with common bead types. To decode nascent proteins that are deposited by PC-PRINT from individual beads, the feasibility of using photocleavable quantum dot codes is demonstrated.
Lim
Mark
M
AmberGen, Watertown, MA 02472, USA. mlim@ambergen.com
Rothschild
Kenneth J
KJ