Pathogen receptor discovery with a microfluidic human membrane protein array Yair Glicka,1,Ya’ara Ben-Aria,1, Nir Draymanb, Michal Pellacha, Gregory Neveuc,d, Jim Boonyaratanakornkitc,d, Dorit Avrahamia, Shirit Einavc,d, Ariella Oppenheimb, and Doron Gerbera,2 aMina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, 5290002, Israel; bFaculty of Medicine, Hebrew University, Jerusalem, 9112001, Israel; cDepartment of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA 94305; and dDepartment of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305 Edited by Stephen R. Quake, Stanford University, Stanford, CA, and approved March 1, 2016 (received for review September 20, 2015) The discovery of how a pathogen invades a cell requires one to and translate into proteins in situ (10, 11). This approach has determine which host cell receptors are exploited. This determina- enabled the study of the Pseudomonas aeruginosa outer mem- tion is a challenging problem because the receptor is invariably a brane protein for immunity (12). membrane protein, which represents an Achilles heel in proteomics. Combining integrated microfluidics with microarrays and We have developed a universal platform for high-throughput ex- in vitro transcription and translation (TNT) systems may over- – pression and interaction studies of membrane proteins by creating a come all of the above mentioned difficulties (Fig. 1A) (13 16). microfluidic-based comprehensive human membrane protein array The integrated microfluidic device allows smart liquid manage- (MPA). The MPA is, to our knowledge, the first of its kind and offers ment in very low volumes, partitioning, and process integration a powerful alternative to conventional proteomics by enabling the (i.e., protein expression, immobilization, and interaction exper- simultaneous study of 2,100 membrane proteins. We characterized iments). Microarray technology provides the means for pro- direct interactions of a whole nonenveloped virus (simian virus 40), gramming thousands of different experiments (17). In vitro TNT as well as those of the hepatitis delta enveloped virus large form expression systems allow protein biosynthesis and are compatible with high throughput (18). Such systems are commercially avail- antigen, with candidate host receptors expressed on the MPA. Se- able and benefit from fast protein expression, low reaction vol- lected newly discovered membrane protein–pathogen interactions umes, and short reaction times and enable expression of synthetic were validated by conventional methods, demonstrating that the proteins with inserted epitope tags. Adding microsomal mem- MPA is an important tool for cellular receptor discovery and for branes enable the correct folding of membrane proteins and understanding pathogen tropism. support posttranslational modifications, such as glycosylation (9). In short, the microfluidic platform facilitates using in vitro pathogen–host interactions | membrane protein array | TNT systems to produce a reliable membrane protein array receptor discovery | integrated microfluidics (MPA) from DNA with high sensitivity, low material and protein consumption, and compatibility with membrane proteins. he human genome contains ∼21,000 distinct protein-coding In this study, we used a microfluidic platform to combine Tgenes (1), out of which ∼5,360 code for membrane proteins microarray technology, cell-free protein expression, and integrated (2). Membrane proteins are critical for many cellular processes, microfluidics, allowing high-throughput screening of pathogens such as signaling, transport, cell–cell communication, and also with a human membrane proteome library. As a proof-of-concept, interaction with pathogens leading to various cellular responses. we screened two pathogens differing in structure and physiology. It is not surprising that 60% of drugs currently in the market The first was simian virus 40 (SV40), a nonenveloped polyomavirus target proteins at the cell surface (3). Mapping molecular inter- containing circular double-stranded DNA, which can induce actions of membrane proteins is, therefore, of utmost importance. membrane invaginations, similarly to other polyomaviruses. It Pathogen–host recognition involves surface interactions regulated causes infections of the kidney and possibly also other tissues by membrane proteins. Many interactions between membrane proteins and pathogens are unknown, partly because of the low Significance sensitivity and limited compatibility of current methodologies with membrane proteins (4). These limitations pose a major obstacle in In this work, we report, to our knowledge, the first in vitro tool understanding pathogen tropism, a public health concern in view for host–pathogen screening that encompasses thousands of of emerging diseases, e.g., severe acute respiratory syndrome and functional insoluble proteins—primarily transmembrane pro- Ebola. There is therefore a need for new approaches that would teins—immobilized within a microfluidic device. We discovered recapitulate the pathogen–host molecular recognition, let alone in previously unknown protein–pathogen interactions, and then the context of intact pathogens. selected interactions were further validated by conventional Mapping protein–protein interaction (PPI) is a major chal- methods. Considering the tremendous difficulty in discovering lenge in proteomics. Many molecular interactions are transient pathogen receptors, this in vitro high-throughput approach is and weak, leading to low yield of bound material and thus de- extremely important and efficient for receptor discovery and manding highly sensitive detection methods. Current methods understanding pathogen tropism, with relevance to emerging for characterizing PPI networks suffer from several basic disad- human diseases. vantages: low sensitivity, leading to high false negative rate; low specificity, leading to high false positive rate (5–7); low coverage Author contributions: Y.G., Y.B.-A., N.D., D.A., S.E., A.O., and D.G. designed research; Y.G., of known interactions; and high variability, even in screens from Y.B.-A., N.D., G.N., J.B., and S.E. performed research; A.O. contributed new reagents/ the same species (4). Protein arrays could potentially override analytic tools; Y.G., Y.B.-A., N.D., M.P., G.N., J.B., D.A., S.E., A.O., and D.G. analyzed data; and Y.G., Y.B.-A., M.P., A.O., and D.G. wrote the paper. such limitations (8), but suffer from a purification bottleneck and limited functionality after deposition (9). These difficulties are The authors declare no conflict of interest. even more pronounced with membrane proteins. Membrane pro- This article is a PNAS Direct Submission. teins are usually in low abundance; in addition, they are in- 1Y.G. and Y.B.-A. contributed equally to this work. compatible with high-throughput methods (e.g., yeast two-hybrid) 2To whom correspondence should be addressed. E-mail: [email protected]. and are difficult to purify in functional form (e.g., protein ar- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. rays). One partial solution to these obstacles was to print DNA 1073/pnas.1518698113/-/DCSupplemental. 4344–4349 | PNAS | April 19, 2016 | vol. 113 | no. 16 www.pnas.org/cgi/doi/10.1073/pnas.1518698113 Downloaded by guest on September 24, 2021 Fig. 1. Membrane protein array generated by integrated microfluidic platform. (A) An integrated microfluidics platform (Left) was used for on-chip ex- pression of membrane proteins, to serve as “baits” for protein interactions or modifications (29). The device consists of two polydimethylsiloxane (PDMS) layers, a flow layer with 64 × 64 unit cells array (gray), and a control layer with micromechanical valves (colored) that manipulate the flow of fluids in the experiment (Center). The sandwich valves (pink) separate neighboring unit cells; the neck valves (green) divide each unit cell into a DNA compartment and a reaction compartment. The button valves (blue) enable surface patterning to promote binding of proteins to an antibody surface. The button valves serve as mechanical traps of molecular interactions (MITOMI) and allow measurement at equilibrium concentration. MITOMI increases the sensitivity of the system, facilitating detection of weak and transient interactions (SI Appendix, Fig. S6). Combining the microfluidic platform with microarray technology enables BIOPHYSICS AND programming of the device with up to several thousand spotted genes (Right). Using assembly PCR (SI Appendix, Fig. S1), we added c-Myc (N-terminal) and COMPUTATIONAL BIOLOGY His6 (C-terminal) tags to the ORFs, creating synthetic genes. On-chip in vitro protein expression, following the synthetic gene programming, combined with the corresponding antibody surface patterning, facilitates the self-assembly of a MPA using cell-free TNT (rabbit reticulocyte). The immobilized bait proteins are labeled with fluorescent antibodies and quantified by using a microarray scanner. Expressed proteins form a green circle below the button valve (Right). (B) We expressed 2,686 different human membrane proteins on chip. The z axis represents the average expression level (n = 4); x and y axes represent the position on-chip in the 2D protein array. Membrane proteins were immobilized on the surface by their C terminus (B) or N terminus (C). Unspotted (red) chambers were used as negative controls. Levels of expression