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Photocleavage-based affinity purification and printing of cell-free expressed proteins: application to proteome microarrays.

ABSTRACT: 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.

SUBMITTER: Lim M

PROVIDER: S-EPMC2784287 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Photocleavage-based affinity purification and printing of cell-free expressed proteins: application to proteome microarrays.

Lim Mark M Rothschild Kenneth J KJ

Analytical biochemistry 20080812 1

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. ...[more]

PMID: 18762158

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Project description:Multiplex serological immunoassays, such as implemented on microarray or microsphere-based platforms, provide greater information content and higher throughput, while lowering the cost and blood volume required. These features are particularly attractive in pediatric food allergy testing to facilitate high throughput multi-allergen analysis from finger- or heel-stick collected blood. However, the miniaturization and microfluidics necessary for creating multiplex assays make them highly susceptible to the "matrix effect" caused by interference from non-target agents in serum and other biofluids. Such interference can result in lower sensitivity, specificity, reproducibility and quantitative accuracy. These problems have in large part prevented wide-spread implementation of multiplex immunoassays in clinical laboratories. We report the development of a novel method to eliminate the matrix effect by utilizing photocleavable capture antibodies to purify and concentrate blood-based biomarkers (a process termed PC-PURE) prior to detection in a multiplex immunoassay. To evaluate this approach, it was applied to blood-based allergy testing. Patient total IgE was purified and enriched using PC-PURE followed by multiplex microsphere-based detection of allergen-specific IgEs (termed the AllerBead assay). AllerBead was formatted to detect the eight most common pediatric food allergens: milk, soy, wheat, egg, peanuts, tree nuts, fin fish and shellfish, which account for >90% of all pediatric food allergies. 205 serum samples obtained from Boston Children's Hospital were evaluated. When PC-PURE was employed with AllerBead, excellent agreement was obtained with the standard, non-multiplex, ImmunoCAP® assay (average sensitivity above published negative predictive cutoffs = 96% and average Pearson r = 0.90; average specificity = 97%). In contrast, poor ImmunoCAP®-correlation was observed when PC-PURE was not utilized (average sensitivity above published negative predictive cutoffs = 59% and average Pearson r = 0.61; average specificity = 97%). This approach should be adaptable to improve a wide range of multiplex immunoassays such as in cancer, infectious disease and autoimmune disease.

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Dataset Information

Photocleavage-based affinity purification and printing of cell-free expressed proteins: application to proteome microarrays.

Publications

Photocleavage-based affinity purification and printing of cell-free expressed proteins: application to proteome microarrays.

Similar Datasets

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