An in vitro microfluidic approach to generating protein-interaction networks

Abstract

We developed an in vitro protein expression and interaction analysis platform based on a highly parallel and sensitive microfluidic affinity assay, and used it for 14,792 on-chip experiments, which exhaustively measured the protein-protein interactions of 43 Streptococcus pneumoniae proteins in quadruplicate. The resulting network of 157 interactions was denser than expected based on known networks. Analysis of the network revealed previously undescribed physical interactions among members of some biochemical pathways.

Figure 1

Experimental design. (a) Graphical representation of expression template design. Both bait and prey DNA templates include a T7 promoter, ribosome binding site (RBS), poly(A) and T7 terminator. The bait library includes a c-myc tag for measuring expression and a T7 tag for protein pull-down. The prey library includes a His tag for detecting interactions. (b) Schematic of the device layout. Each unit cell is controlled by three micromechanical valves as well as a ‘button‘ membrane used for surface derivatization and MITOMI. Bait and Prey DNA templates are aligned under the chip’s DNA chambers in which proteins are expressed. Pull-down antibody is deposited under the button valve. Unit cell size is 281.25 mm by 562.5 mm and an area of 158,203 mm². Average cell height is 10 mm, and average cell volume is less than 1 nl. Unit cell density is 632 cm⁻². (c) Image of a typical bait (green) and prey (red) interaction on chip. Interaction is in orange owing to overlay of the two channels. Scale bar, 100 mm.

Figure 2

On-chip protein expression. (a) Protein expression levels (in arbitrary fluorescence units) of 43 different S. pneumoniae proteins were measured with Cy3-labeled antibody to c-myc. Error bars, s.e.m. n = 4. (b) Expression levels as a function of protein size. (c) Variation in expression levels (s.d.) as a function of protein size. (d,e) Distribution histograms of protein expression levels (d) and protein size (e) in the 43-protein set.

Figure 3

Analysis of protein-protein interactions. (a) A three-dimensional plot of protein interactions as a function of bait and prey expression. Interactions (in arbitrary fluorescence units) are represented by spheres: specific or bidirectional interactions (blue), nonspecific or unidirectional interactions (gold), ’confirmed interactions‘ from the SwissProt database detected by PING (red) and SwissProt interactions that PING failed to detect (cyan). The cutoff was determined as 2 s.d. above the average signal resulting from anti-HIS binding to the corresponding bait-only expressing cells. (b) Verification of protein interactions by co-immunoprecipitation experiments. Six pairs of known interactors, six pairs of previously unknown interactors and one pair of proteins that do not interact were tested. Bait and prey protein pairs were co-expressed in vitro and then co-immunoprecipitated using Nickel beads. Prey levels were detected with Alexa 647-labeled antibody to the T7 tag. The data were plotted as net signal, in units of s.d. of the background.

Figure 4

S. pneumoniae interaction network represented by a hairball graph created with Cytoscape 2.4. Blue edges represent interactions found by PING screens. Red edges represent confirmed interactions from the Swissprot database. Heat shock proteins were omitted for clarity.