Facile Protein Immobilization Using Engineered Surface-ActiveBiofilm ProteinsDanielle M. Williams,† Gilad Kaufman,‡ Hadi Izadi,‡ Abigail E. Gahm,§ Sarah M. Prophet,†

Kyle T. Vanderlick,‡ Chinedum O. Osuji,‡ and Lynne Regan*,†,⊥,#

†Departments of Molecular Biophysics and Biochemistry, ‡Chemical and Environmental Engineering, §Molecular, Cellular, andDevelopmental Biology, ⊥Chemistry, and #The Integrated Graduate Program in Physical and Engineering Biology, Yale University,New Haven, Connecticut 06511, United States

*S Supporting Information

ABSTRACT: Immobilization of enzymes and other biomolecules tosurfaces is critically important for biotechnology, with important appli-cations in sensing and controlled delivery of molecular species for ana-lytical or biomedical purposes. The presentation of protein recognitionelements in a way that avoids denaturation and nonspecific interactionswhile maintaining the accessibility of the active site is a challenge forwhich no general solution has been found. Here we present a robust,facile method for immobilization of any protein to a surface usingengineered protein building blocks. By functionalizing an interfacialprotein, BslA, with peptides (SpyTag and SnoopTag) that spontaneouslyreact with their cognate protein partners (SpyCatcher and SnoopCatcher),we are able to create patterned surfaces of protein monolayers displayingreactive tags. We demonstrate that these surfaces can be functionalizedrapidly, spontaneously, and specifically with proteins of interest attachedto SpyCatcher or SnoopCatcher. This method both protects the surface from nonspecific adsorption and also presents therecognition element in a uniform, active conformation. We envision that this method will have widespread applications, includingimmobilization of therapeutically relevant proteins for diagnostic applications.

KEYWORDS: protein engineering, self-assembly, hydrophobin, modular, interfacial protein, protein immobilization

■ INTRODUCTION

The immobilization of biomolecules on solid supports hasmany applications in biotechnology and biomedicine, includingmicroarrays of nucleic acids (or proteins) and a multitude ofELISA-like biosensors.1 Surface immobilization is convenientfor the exposure to analyte, washing, and detection steps of theprocess. The ability to site-specifically array proteins ontosurfaces allows for the high-throughput detection of analytes.2

A typical biosensor requires immobilization of the recognitionelement, which is often a protein.3−6 Achieving a consistentpresentation of the protein recognition element on a surfacewhile simultaneously avoiding undesirable, and potentially dena-turing, interactions of that protein with the surface is still anunsolved problem.6 Accomplishing a consistent presentation ofa native protein on a surface increases the accessibility of thatprotein to the analyte and maximizes the number of nativeproteins in a given area, both of which increase the sensitivityof analyte detection. Nonspecific, noncovalent sticking of therecognition element to a surface, such as the polystyrene of amicrotiter plate, is often sufficient for laboratory applications.In many settings, however, the greatest possible sensitivityand specificity of detection is required, and a convenient way toconsistently present the maximum amount of functional recogni-tion element in a given surface area is desirable. Several different

approaches have been taken to address this important issue, buta straightforward and widely applicable strategy has not yetbeen established.7−11

Another approach that has been taken is to coat surfaceswith bioaffinity reagents. For example, a surface coated withavidin or streptavidin will bind a biotinylated molecule, and asurface coated with Ni-NTA will bind a hexahistidine-taggedprotein.12−14 Both of these methods involve a noncovalentinteraction of the recognition element to a functionalizedsurface. The efficacy of the NTA method is hindered by the lowbinding affinity of the hexahistidine tag to Ni2+-NTA, whichoften does not survive the requisite multiple washing steps.In addition, for some proteins, issues with metal-dependent,nonspecific protein adsorption to the surface are also aproblem.15 Although the interaction of streptavidin (or avidin)with biotin is also noncovalent, it is extremely high affinity.A main issue with the use of streptavidin is that there are fourpotential binding sites for biotin per streptavidin molecule,which can result in heterogeneity of the immobilization andpresentation.

Received: March 30, 2018Accepted: April 19, 2018Published: May 31, 2018

Letter

www.acsanm.orgCite This: ACS Appl. Nano Mater. 2018, 1, 2483−2488

© 2018 American Chemical Society 2483 DOI: 10.1021/acsanm.8b00520ACS Appl. Nano Mater. 2018, 1, 2483−2488

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Covalent interactions provide a more stable method ofattachment. A common covalent attachment method is throughthe use of “click” chemistry, a term that encompasses manyreactions, several of which take advantage of alkyne chemistry.Click reactions can be rapid, specific, and work well underaqueous conditions. However, they typically require theincorporation of a nonnatural amino acid (such as one withan azido-containing side chain) into the protein to beimmobilized, to react with an alkyne on the surface. Achievinghigh-efficiency incorporation of nonnatural amino acidsremains an area of active investigation.16 Another method ofchemoligation is to coat a surface with a molecule terminatedin an N-hydroxysuccinimide ester,17,18 which can react withthe primary amine groups of the Lys residue on the surface ofa protein to form an amide bond. A significant limitation ofthis method is the competing reaction of ester hydrolysis inaqueous solution, resulting in relatively low yields of proteinattachment.12 Additionally, Lys residues are abundant onprotein surfaces, so binding is rarely site-specific binding, andthe resultant surface has significant conformational hetero-geneity.15 Native peptide ligation (NPL) and numerousimmobilization techniques related to protein splicing havebeen developed as derivatives of NPL, with one notablemethod being expressed protein ligation (EPL). These methodsare advantageous because they result in the formation of a

covalent bond and are “traceless”, but many reported proce-dures require lengthy incubation times that can be up to severaldays.12

Here we present a straightforward, specific, and scalablemethod to covalently immobilize proteins of interest to asurface. The method successfully exploits the unique physicaland chemical properties of natural proteins: BslA (Figure 1a),which self-assembles to form a monolayer at a hydrophobic/hydrophilic interface,19 and engineered streptococcal surfaceproteins,20−22 which spontaneously form a covalent isopeptidebond between Lys and Asp/Asn side chains on two differentpolypeptides. The availability of two different protein pairs,SpyCatcher and SpyTag and also SnoopCatcher and SnoopTag(Figure 1c,d), which do not cross-react, provides a route for thesimultaneous display of different protein recognition elements.By fusing BslA (16 kDa) to SpyTag (13-residue peptide) andSnoopTag (12-residue peptide) and fluorescent proteins toSpyCatcher (12 kDa) and SnoopCatcher (13 kDa), we createdreactive pairs of proteins for use in our method (Figure 1b).Key features of this method are that all of the components areexpressed recombinantly and react spontaneously with highefficiency. We anticipate that a multiplexed, spatially distinctdisplay of several recognition elements, as demonstrated in thiswork, will facilitate single-sample multianalyte detection.

Figure 1. Cartoon and ribbon representations of the protein building blocks. (a) Ribbon (from PDB 4BHU, left) and cartoon representation (right)of the structure of BslA. The orange coloring indicates the hydrophobic N-terminal region, and the blue coloring indicates the hydrophilicC-terminal region. (b) Cartoon representations of BslA and fluorescent fusion proteins. SpyTag (teal triangle) and SnoopTag (purple triangle) areattached to the C terminus of BslA (orange and blue ovals). SpyCatcher (maroon crown) and SnoopCatcher (gold crown) are attached to theC terminus of eGFP (green starburst) and mCherry (red starburst), respectively. (c) Ribbon (from PDB 4MLI) and cartoon representations of theSpyCatcher protein (maroon crown) and SpyTag (teal triangle). The formation of a covalent bond between the side chains of Lys on SpyCatcherand Asp on SpyTag is shown. (d) Cartoon representation of SnoopCatcher (gold crown) and SnoopTag (purple triangle). The formation of acovalent bond between the side chains of Asn on SnoopCatcher and Lys on SnoopTag is shown.

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■ RESULTS AND DISCUSSIONWild-type (wt)BslA self-assembles at air−water interfaces toform robust monolayers, the properties of which have been well

characterized.19,23−26 For the studies reported here, we used aLangmuir−Blodgett (LB) apparatus to form consistent, well-packed monolayers of BslA. We previously showed that theaddition of the 13-residue peptide SpyTag to the C terminus ofBslA does not significantly perturb the formation of the BslAmonolayer.27 In the functionalization studies presented here,we used mixtures of 25% BslA−SpyTag/75% wt BslA and 25%BslA−SnoopTag/75% wt BslA. We chose mixtures of taggedand wt BslA, as opposed to using 100% BslA−SpyTag and100% BslA−SnoopTag, to decrease the steric interference betweenmolecules attached to the monolayer via BslA’s C-terminalpeptide. We characterized the behavior of such monolayersby measuring surface pressure−area isotherms using an LBapparatus (Figure 2). The comparable collapse pressures and

Figure 2. Surface pressure−area isotherms of BslA constructs. Datawere obtained using a LB apparatus. For each protein, a surfacepressure versus area isotherm was measured in three independentexperiments. Data from individual experiments are shown in black(circles, squares, and diamonds), and the average of the threemeasurements is shown in colored triangles (100% wt BslA, orange;25% BslA−SpyTag/75% wt BslA, teal; 25% BslA−SnoopTag/75% wtBslA, purple). The different protein monolayers all exhibit a similarcollapse pressure of ∼65 mN/m. We calculate the average area permolecule at 23 mN/m, which corresponds to the maximum surfacepressure achievable before exerting mechanical compression force, tobe 656 Å2 for wt BslA (a), 753 Å2 for 25% BslA−SpyTag/75% wt BslA(b), and 679 Å2 for 25% BslA−Snooptag/75% wt BslA (c). Thevariability associated with different measurements of the samemonolayer was calculated as the standard deviation of each trial, andall were minimal (around ±1%). Much more variability is associatedwith determination of the protein concentration (around ±20%).

Figure 3. Schematic of the different processes by which slides werepatterned with FOTS, deposited with BslA protein, and probed withfluorescent proteins. Cartoons are not to scale. (a) PDMS micropillarstamps (dark-gray apparatus with pillars) were incubated with a FOTSsolution (orange) before being wicked away with a tissue, yielding an“inked” stamp. The stamp was placed on top of a clean glass slide(small dark-gray rectangle) with a 20 g weight on top. The weight andstamp were removed, leaving behind a glass slide printed with ahexagonal pattern of circular, hydrophobic FOTS spots. (b) 25%BslA−SpyTag/75% wt BslA or 25% BslA−SnoopTag/75% wt BslA(orange and blue ovals) were injected and allowed to equilibrate to theair−water interface of a LB trough (light-gray apparatus). Afterequilibration, the barriers were compressed to a surface pressure of23 mN/m, forming a protein monolayer at the air−water interface.The patterned slides prepared from (a) were lowered to make contactwith the protein monolayer using a LS apparatus (light-gray rectangleabove the LB trough). After making contact with the monolayer, thehydrophobic ends of 25% BslA−SpyTag/75% wt BslA or 25% BslA−SnoopTag/75% wt BslA were transferred to the slide at the sites of thehydrophobic spots, resulting in a patterned slide displaying a pro-tein monolayer. The slides were stored in DI water until furtheruse. (c) Patterned slides displaying a protein monolayer prepared inpart b were incubated with a solution of 20−50 μM GFP−SpyCatcheror mCherry−SnoopCatcher (middle cartoon). Excess fluorescentproteins that did not bind to BslA−SpyTag or BslA−SnoopTagproteins were washed away with DI water to yield slides withfluorescently labeled circular spots (right cartoons). After rinsing, theslides were wicked dry with a tissue and imaged using fluorescencemicroscopy.

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mean molecular areas make it clear that the behavior of thesemixed monolayers is very similar to that of 100% wt BslA indi-cating that the C-terminal fusions to the SpyTag and SnoopTagpeptides cause little perturbation to the monolayer.BslA-based protein monolayers were transferred from the

air−water interface to a hydrophobic/water surface using aLangmuir−Schaefer (LS) adaptor. We first used microcontactprinting28 to create slides with a distinct pattern of individualhydrophobic spots on a glass surface (Figure 3a). The hydro-phobic spots were created by microcontact printing of trichloro-(1H,1H,2H,2H-perfluorooctyl)silane (FOTS) suspended in etha-nol using a poly(dimethlysiloxane) (PDMS) stamp. The remainderof the surface of the glass slide was untreated. Protein monolayerswere formed using an LB apparatus and compressed to a surfacepressure of 23 mN/m. This value was chosen based on priorstudies that show that it results in the formation of monolayersthat are reliably free of significant distortion.26 The monolayerswere then transferred to the patterned glass slide using an LSattachment (Figure 3b).In preliminary experiments, we tested the ability of a mono-

layer of wt BslA on the hydrophobic surface to prevent non-specific adsorption of fluorescent proteins to glass. In the absenceof a BslA coating, the fluorescent protein fusions readily adsorbnonspecifically to the hydrophobic surface (Figure S2). By con-trast, such nonspecific binding is effectively eliminated whenthe surface bears a monolayer of BslA. This is evident from thereduction of fluorescence in the area of the hydrophobic spotrelative to that of nonspecific binding to the glass slide (Figure S2).The data are noteworthy because they demonstrate that, inaddition to providing a novel means to attach a protein of inter-est to a surface, the BslA coating can also eliminate nonspecificbinding of proteins of interest to that surface. In the contextof sensing, this is expected to reduce false negatives, i.e., to

increase the confidence with which one can conclude that aspecies of interest is not present.Three different surfaces were created and tested: 25% BslA−

SpyTag/75% wt BslA, 25% BslA−SnoopTag/75% wt BslAand 100% wt BslA. Surfaces were probed with either eGFP−SpyCatcher or mCherry−SnoopCatcher, rinsed with deionized(DI) water (Figure 3c), and imaged using fluorescence micros-copy (Figure 4). From these images, it is clear that eGFP−SpyCatcher only reacts with and labels surfaces that containBslA−SpyTag and mCherry−SnoopCatcher only reacts withand labels surfaces that contain BslA−SnoopTag. NeithereGFP−SpyCatcher nor mCherry−SnoopCatcher binds to thehydrophobic surface coated with BslA or with the noncognateBslA−SpyTag or BslA−SnoopTag (Figure 4a,c). Indeed, the BslAcoatings reduce background binding to less than the backgroundbinding of the fluorescent protein to the uncoated glass slide.These observations are shown quantitatively in plots comparingthe signal to the background fluorescence intensity for eachsurface after probing (Figure 4b,d). The surfaces in the experi-ments shown were incubated with fluorescent protein fusionsfor 10 min, but we observed no change in fluorescence from theovernight incubations (data not shown). Thus, this strategy ofattaching proteins to surfaces is both specific with respect torequiring a cognate SpyTag/SpyCatcher or SnoopTag/Snoop-Catcher pair and also essentially eliminates background bindingto the hydrophobic surface.Preliminary experiments suggest that the scope of this method

could be broadened by changing the component fused to BslA(for example, SpyCatcher rather than SpyTag) and thecomponent fused to the protein to be immobilized (SpyTagrather than SpyCatcher). One can readily envision the use ofthe immobilization strategy presented here for practical bio-sensing applications, for example, by immobilizing a protein

Figure 4.Microscope images and quantitative analysis of fluorescence. All scale bars are 50 μm. (a) Fluorescence microscopy images of slides printedwith a pattern of FOTS, displaying a monolayer of 25% BslA−SpyTag/75% wt BslA (left), 25% BslA−SnoopTag/75% wt BslA (middle), or 100% wtBslA (right) were incubated with GFP−SpyCatcher. The image has been falsely colored to show eGFP fluorescence as green. (b) Fluorescence intensityprofiles over four spots, from the images in part a: slides patterned with 100% wt BslA (orange circles), 25% BslA−SpyTag/75% wt BslA (teal triangles),and 25% BslA−SnoopTag/75% wt BslA (purple squares) probed with eGFP−SpyCatcher. (c) Fluorescence microscopy images of slides printed with apattern of FOTS displaying a monolayer of 25% BslA−SpyTag/75% wt BslA (left), 25% BslA−SnoopTag/75% wt BslA (middle), or 100% wt BslA(right) were incubated with mCherry−SnoopCatcher. The image has been falsely colored to show the mCherry fluorescence as red. (d) Fluorescenceintensity profiles over four spots, from the images in part c: slides patterned with 100% wt BslA (orange circles), 25% BslA−SpyTag/75% wt BslA(teal triangles), and 25% BslA−SnoopTag/75% wt BslA (purple squares) probed with mCherry−SnoopCatcher.

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against which an organism produces antibodies and thereafterdetecting the presence of those antibodies. Another examplecould be immobilization of a recognition protein that binds to amolecule of interest, which is then detected by a second recog-nition protein, as in a “sandwich ELISA” assay.

■ CONCLUSIONBy exploiting the self-assembling properties of natural proteins,we have created a simple but highly effective method for theimmobilization of recognition elements to a surface. We demon-strated that functionalizing BslA with SpyTag and SnoopTagpeptides does not significantly perturb monolayer formation.In principle, the method presented can be applied for any pro-tein of interest attached to SpyCatcher or SnoopCatcher. Whenprobing surfaces with fluorescent protein fusions, we did notobserve any cross-reactivity of Snoop variants with Spy variantsand additionally saw no nonspecific binding to surfaces depos-ited with 100% wt BslA. Moreover, the BslA coating is effectivein eliminating nonspecific protein surface binding, which couldbe useful for a broad range of other applications. Preliminaryexperiments indicate that it is possible for SpyCatcher/SnoopCatcher to be fused to BslA as opposed to the proteinof interest, which would greatly increase the scope of thismethod. We anticipate the future application of the strategythat we describe to immobilize therapeutically relevant proteinstoward the production of biosensors with increased sensitivityand specificity.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsanm.8b00520.

Experimental procedures, protein and DNA sequences,and associated figures (PDF)

■ AUTHOR INFORMATIONCorresponding Author*E-mail: lynne.regan@yale.edu.ORCIDDanielle M. Williams: 0000-0001-6763-3775Chinedum O. Osuji: 0000-0003-0261-3065Author ContributionsThe manuscript was written through contributions of allauthors. All authors have given approval to the final version ofthe manuscript.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe gratefully acknowledge support from the NSF through theYale Materials Research Science and Engineering Center(Grant MRSEC DMR-1119826) and from the Raymond andBeverly Sackler Institute for Biological, Physical and Engineer-ing Sciences. C.O.O. acknowledges NSF support under GrantCMMI-1246804 and the facilities of the Yale Institute for Nanoand Quantum Engineering (YINQE); L.R. acknowledges supportfrom the NSF (Grant DMR-1307712); D.M.W. acknowledgessupport from the NIH Cellular and Molecular Biology TrainingGrant (Grant CMB TG T32GM007223); S.M.P. receivedfunding from a Yale University fellowship; A.E.G. acknowledgesfunding from the Yale College Dean’s Research Fellowship.

■ ABBREVIATIONS

BslA = biofilm surface layer proteinFOTS = trichloro(1H,1H,2H,2H-perfluorooctyl)silaneGFP = green fluorescent proteinGST = glutathione S transferaseLB = Langmuir−BlodgettLS = Langmuir−SchaeferPDMS = poly(dimethylsiloxane)WT = wild type

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