Self-Assembling Protein Microarrays

Abstract

Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.

Mapping transcription factor interactome networks using HaloTag protein arrays

“…Previous NAPPA technology used an anti-GST antibody to anchor in vitro expressed C-terminal GST fusion proteins to an aminosilanized glass slide (9,10). We modified this original system by incorporating a high-affinity capture tag, the HaloTag (Promega), to immobilize nascent proteins on the array surface (18).…”

“…With conventional protein microarrays it is necessary to purify thousands of in vivo expressed proteins and to spot these purified proteins on a solid surface (5-8). In contrast, in situ synthesis protein microarray technologies simplify protein microarray fabrication by circumventing the steps of in vivo protein expression and purification (9,10,14,15). This streamlining facilitates an increase in the number of target genes that can be assayed, allowing for thousands of protein-encoding plasmids to be spotted at lower cost and in less time.…”

“…This streamlining facilitates an increase in the number of target genes that can be assayed, allowing for thousands of protein-encoding plasmids to be spotted at lower cost and in less time. Such a large scale has been achieved with the nucleic acid programmable protein array (NAPPA) technology (9,10), which uses a cell-free expression system and antibody-based protein capture to create protein microarrays from DNA microarrays. Current NAPPA technology, however, has been hampered by the low efficiency of antibody-based protein capture, which requires large spot sizes, resulting in low spot density (∼2,000 spots per array) (10).…”

An experimental strategy for quantitative analysis of the humoral immune response to prostate cancer antigens using natural protein microarrays

“…Our future validation studies will focus on proteins identified in fresh experiments with the new methods of separation suggested above, since those new methods will provide more precise leads on the candidate proteins. In order to study future candidates efficiently, it may be valuable to have a high-throughput protein expression system, for example using on-chip translation of selected sequences [29]. However, immunoreactivity may only be present in proteins taken directly from cell lines or tumor tissue, so it may be necessary to use proteins purified from those materials.…”

Next generation microfluidic platforms for high-throughput protein biochemistry

“…In 2004 Ramachandran et al developed a method for transforming spotted DNA arrays into protein arrays, termed nucleic acid programmable protein array (NAPPA) [9,10 ]. Expression-ready biotinylated plasmid DNA is co-spotted on a glass substrate together with a biotinylated antibody.…”