Facile Protein Immobilization Using Engineered Surface-Active Biofilm

Publication Date (Web): May 31, 2018. Copyright © 2018 American ... we are able to create patterned surfaces of protein monolayers displaying reactiv...
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Letter Cite This: ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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Facile Protein Immobilization Using Engineered Surface-Active Biofilm Proteins Danielle 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, and Developmental 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 to surfaces is critically important for biotechnology, with important applications in sensing and controlled delivery of molecular species for analytical or biomedical purposes. The presentation of protein recognition elements in a way that avoids denaturation and nonspecific interactions while maintaining the accessibility of the active site is a challenge for which no general solution has been found. Here we present a robust, facile method for immobilization of any protein to a surface using engineered protein building blocks. By functionalizing an interfacial protein, BslA, with peptides (SpyTag and SnoopTag) that spontaneously react with their cognate protein partners (SpyCatcher and SnoopCatcher), we are able to create patterned surfaces of protein monolayers displaying reactive tags. We demonstrate that these surfaces can be functionalized rapidly, spontaneously, and specifically with proteins of interest attached to SpyCatcher or SnoopCatcher. This method both protects the surface from nonspecific adsorption and also presents the recognition element in a uniform, active conformation. We envision that this method will have widespread applications, including immobilization of therapeutically relevant proteins for diagnostic applications. KEYWORDS: protein engineering, self-assembly, hydrophobin, modular, interfacial protein, protein immobilization

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INTRODUCTION The immobilization of biomolecules on solid supports has many applications in biotechnology and biomedicine, including microarrays of nucleic acids (or proteins) and a multitude of ELISA-like biosensors.1 Surface immobilization is convenient for the exposure to analyte, washing, and detection steps of the process. The ability to site-specifically array proteins onto surfaces allows for the high-throughput detection of analytes.2 A typical biosensor requires immobilization of the recognition element, which is often a protein.3−6 Achieving a consistent presentation of the protein recognition element on a surface while simultaneously avoiding undesirable, and potentially denaturing, interactions of that protein with the surface is still an unsolved problem.6 Accomplishing a consistent presentation of a native protein on a surface increases the accessibility of that protein to the analyte and maximizes the number of native proteins in a given area, both of which increase the sensitivity of analyte detection. Nonspecific, noncovalent sticking of the recognition element to a surface, such as the polystyrene of a microtiter plate, is often sufficient for laboratory applications. In many settings, however, the greatest possible sensitivity and specificity of detection is required, and a convenient way to consistently present the maximum amount of functional recognition element in a given surface area is desirable. Several different © XXXX American Chemical Society

approaches have been taken to address this important issue, but a straightforward and widely applicable strategy has not yet been established.7−11 Another approach that has been taken is to coat surfaces with bioaffinity reagents. For example, a surface coated with avidin or streptavidin will bind a biotinylated molecule, and a surface coated with Ni-NTA will bind a hexahistidine-tagged protein.12−14 Both of these methods involve a noncovalent interaction of the recognition element to a functionalized surface. The efficacy of the NTA method is hindered by the low binding affinity of the hexahistidine tag to Ni2+-NTA, which often 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 a problem.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 four potential binding sites for biotin per streptavidin molecule, which can result in heterogeneity of the immobilization and presentation. Received: March 30, 2018 Accepted: April 19, 2018

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DOI: 10.1021/acsanm.8b00520 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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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 hydrophilic C-terminal region. (b) Cartoon representations of BslA and fluorescent fusion proteins. SpyTag (teal triangle) and SnoopTag (purple triangle) are attached to the C terminus of BslA (orange and blue ovals). SpyCatcher (maroon crown) and SnoopCatcher (gold crown) are attached to the C terminus of eGFP (green starburst) and mCherry (red starburst), respectively. (c) Ribbon (from PDB 4MLI) and cartoon representations of the SpyCatcher protein (maroon crown) and SpyTag (teal triangle). The formation of a covalent bond between the side chains of Lys on SpyCatcher and Asp on SpyTag is shown. (d) Cartoon representation of SnoopCatcher (gold crown) and SnoopTag (purple triangle). The formation of a covalent bond between the side chains of Asn on SnoopCatcher and Lys on SnoopTag is shown.

covalent bond and are “traceless”, but many reported procedures require lengthy incubation times that can be up to several days.12 Here we present a straightforward, specific, and scalable method to covalently immobilize proteins of interest to a surface. The method successfully exploits the unique physical and chemical properties of natural proteins: BslA (Figure 1a), which self-assembles to form a monolayer at a hydrophobic/ hydrophilic interface,19 and engineered streptococcal surface proteins,20−22 which spontaneously form a covalent isopeptide bond between Lys and Asp/Asn side chains on two different polypeptides. 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 the simultaneous display of different protein recognition elements. By fusing BslA (16 kDa) to SpyTag (13-residue peptide) and SnoopTag (12-residue peptide) and fluorescent proteins to SpyCatcher (12 kDa) and SnoopCatcher (13 kDa), we created reactive pairs of proteins for use in our method (Figure 1b). Key features of this method are that all of the components are expressed recombinantly and react spontaneously with high efficiency. We anticipate that a multiplexed, spatially distinct display of several recognition elements, as demonstrated in this work, will facilitate single-sample multianalyte detection.

Covalent interactions provide a more stable method of attachment. A common covalent attachment method is through the use of “click” chemistry, a term that encompasses many reactions, several of which take advantage of alkyne chemistry. Click reactions can be rapid, specific, and work well under aqueous conditions. However, they typically require the incorporation of a nonnatural amino acid (such as one with an azido-containing side chain) into the protein to be immobilized, to react with an alkyne on the surface. Achieving high-efficiency incorporation of nonnatural amino acids remains an area of active investigation.16 Another method of chemoligation is to coat a surface with a molecule terminated in an N-hydroxysuccinimide ester,17,18 which can react with the primary amine groups of the Lys residue on the surface of a protein to form an amide bond. A significant limitation of this method is the competing reaction of ester hydrolysis in aqueous solution, resulting in relatively low yields of protein attachment.12 Additionally, Lys residues are abundant on protein surfaces, so binding is rarely site-specific binding, and the resultant surface has significant conformational heterogeneity.15 Native peptide ligation (NPL) and numerous immobilization techniques related to protein splicing have been developed as derivatives of NPL, with one notable method being expressed protein ligation (EPL). These methods are advantageous because they result in the formation of a B

DOI: 10.1021/acsanm.8b00520 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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ACS Applied Nano Materials

Figure 3. Schematic of the different processes by which slides were patterned with FOTS, deposited with BslA protein, and probed with fluorescent proteins. Cartoons are not to scale. (a) PDMS micropillar stamps (dark-gray apparatus with pillars) were incubated with a FOTS solution (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 and stamp were removed, leaving behind a glass slide printed with a hexagonal 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 the air−water interface of a LB trough (light-gray apparatus). After equilibration, the barriers were compressed to a surface pressure of 23 mN/m, forming a protein monolayer at the air−water interface. The patterned slides prepared from (a) were lowered to make contact with the protein monolayer using a LS apparatus (light-gray rectangle above the LB trough). After making contact with the monolayer, the hydrophobic ends of 25% BslA−SpyTag/75% wt BslA or 25% BslA− SnoopTag/75% wt BslA were transferred to the slide at the sites of the hydrophobic spots, resulting in a patterned slide displaying a protein monolayer. The slides were stored in DI water until further use. (c) Patterned slides displaying a protein monolayer prepared in part b were incubated with a solution of 20−50 μM GFP−SpyCatcher or mCherry−SnoopCatcher (middle cartoon). Excess fluorescent proteins that did not bind to BslA−SpyTag or BslA−SnoopTag proteins were washed away with DI water to yield slides with fluorescently labeled circular spots (right cartoons). After rinsing, the slides were wicked dry with a tissue and imaged using fluorescence microscopy.

Figure 2. Surface pressure−area isotherms of BslA constructs. Data were obtained using a LB apparatus. For each protein, a surface pressure versus area isotherm was measured in three independent experiments. Data from individual experiments are shown in black (circles, squares, and diamonds), and the average of the three measurements is shown in colored triangles (100% wt BslA, orange; 25% BslA−SpyTag/75% wt BslA, teal; 25% BslA−SnoopTag/75% wt BslA, purple). The different protein monolayers all exhibit a similar collapse pressure of ∼65 mN/m. We calculate the average area per molecule at 23 mN/m, which corresponds to the maximum surface pressure achievable before exerting mechanical compression force, to be 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). The variability associated with different measurements of the same monolayer was calculated as the standard deviation of each trial, and all were minimal (around ±1%). Much more variability is associated with determination of the protein concentration (around ±20%).

characterized.19,23−26 For the studies reported here, we used a Langmuir−Blodgett (LB) apparatus to form consistent, wellpacked monolayers of BslA. We previously showed that the addition of the 13-residue peptide SpyTag to the C terminus of BslA does not significantly perturb the formation of the BslA monolayer.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 tagged and wt BslA, as opposed to using 100% BslA−SpyTag and 100% BslA−SnoopTag, to decrease the steric interference between molecules attached to the monolayer via BslA’s C-terminal peptide. We characterized the behavior of such monolayers by measuring surface pressure−area isotherms using an LB apparatus (Figure 2). The comparable collapse pressures and

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RESULTS AND DISCUSSION Wild-type (wt)BslA self-assembles at air−water interfaces to form robust monolayers, the properties of which have been well C

DOI: 10.1021/acsanm.8b00520 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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Figure 4. Microscope images and quantitative analysis of fluorescence. All scale bars are 50 μm. (a) Fluorescence microscopy images of slides printed with a pattern 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 GFP−SpyCatcher. The image has been falsely colored to show eGFP fluorescence as green. (b) Fluorescence intensity profiles 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 a pattern 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) Fluorescence intensity 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.

mean molecular areas make it clear that the behavior of these mixed monolayers is very similar to that of 100% wt BslA indicating that the C-terminal fusions to the SpyTag and SnoopTag peptides cause little perturbation to the monolayer. BslA-based protein monolayers were transferred from the air−water interface to a hydrophobic/water surface using a Langmuir−Schaefer (LS) adaptor. We first used microcontact printing28 to create slides with a distinct pattern of individual hydrophobic spots on a glass surface (Figure 3a). The hydrophobic spots were created by microcontact printing of trichloro(1H,1H,2H,2H-perfluorooctyl)silane (FOTS) suspended in ethanol using a poly(dimethlysiloxane) (PDMS) stamp. The remainder of the surface of the glass slide was untreated. Protein monolayers were formed using an LB apparatus and compressed to a surface pressure of 23 mN/m. This value was chosen based on prior studies that show that it results in the formation of monolayers that are reliably free of significant distortion.26 The monolayers were then transferred to the patterned glass slide using an LS attachment (Figure 3b). In preliminary experiments, we tested the ability of a monolayer of wt BslA on the hydrophobic surface to prevent nonspecific adsorption of fluorescent proteins to glass. In the absence of a BslA coating, the fluorescent protein fusions readily adsorb nonspecifically to the hydrophobic surface (Figure S2). By contrast, such nonspecific binding is effectively eliminated when the surface bears a monolayer of BslA. This is evident from the reduction of fluorescence in the area of the hydrophobic spot relative to that of nonspecific binding to the glass slide (Figure S2). The data are noteworthy because they demonstrate that, in addition to providing a novel means to attach a protein of interest to a surface, the BslA coating can also eliminate nonspecific binding of proteins of interest to that surface. In the context of sensing, this is expected to reduce false negatives, i.e., to

increase the confidence with which one can conclude that a species of interest is not present. Three different surfaces were created and tested: 25% BslA− SpyTag/75% wt BslA, 25% BslA−SnoopTag/75% wt BslA and 100% wt BslA. Surfaces were probed with either eGFP− SpyCatcher or mCherry−SnoopCatcher, rinsed with deionized (DI) water (Figure 3c), and imaged using fluorescence microscopy (Figure 4). From these images, it is clear that eGFP− SpyCatcher only reacts with and labels surfaces that contain BslA−SpyTag and mCherry−SnoopCatcher only reacts with and labels surfaces that contain BslA−SnoopTag. Neither eGFP−SpyCatcher nor mCherry−SnoopCatcher binds to the hydrophobic surface coated with BslA or with the noncognate BslA−SpyTag or BslA−SnoopTag (Figure 4a,c). Indeed, the BslA coatings reduce background binding to less than the background binding of the fluorescent protein to the uncoated glass slide. These observations are shown quantitatively in plots comparing the signal to the background fluorescence intensity for each surface after probing (Figure 4b,d). The surfaces in the experiments shown were incubated with fluorescent protein fusions for 10 min, but we observed no change in fluorescence from the overnight incubations (data not shown). Thus, this strategy of attaching proteins to surfaces is both specific with respect to requiring a cognate SpyTag/SpyCatcher or SnoopTag/SnoopCatcher pair and also essentially eliminates background binding to 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 the component fused to the protein to be immobilized (SpyTag rather than SpyCatcher). One can readily envision the use of the immobilization strategy presented here for practical biosensing applications, for example, by immobilizing a protein D

DOI: 10.1021/acsanm.8b00520 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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against which an organism produces antibodies and thereafter detecting the presence of those antibodies. Another example could be immobilization of a recognition protein that binds to a molecule of interest, which is then detected by a second recognition protein, as in a “sandwich ELISA” assay.

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CONCLUSION By exploiting the self-assembling properties of natural proteins, we have created a simple but highly effective method for the immobilization of recognition elements to a surface. We demonstrated that functionalizing BslA with SpyTag and SnoopTag peptides does not significantly perturb monolayer formation. In principle, the method presented can be applied for any protein of interest attached to SpyCatcher or SnoopCatcher. When probing surfaces with fluorescent protein fusions, we did not observe any cross-reactivity of Snoop variants with Spy variants and additionally saw no nonspecific binding to surfaces deposited with 100% wt BslA. Moreover, the BslA coating is effective in eliminating nonspecific protein surface binding, which could be useful for a broad range of other applications. Preliminary experiments indicate that it is possible for SpyCatcher/ SnoopCatcher to be fused to BslA as opposed to the protein of interest, which would greatly increase the scope of this method. We anticipate the future application of the strategy that we describe to immobilize therapeutically relevant proteins toward the production of biosensors with increased sensitivity and specificity.

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ABBREVIATIONS BslA = biofilm surface layer protein FOTS = trichloro(1H,1H,2H,2H-perfluorooctyl)silane GFP = green fluorescent protein GST = glutathione S transferase LB = Langmuir−Blodgett LS = Langmuir−Schaefer PDMS = poly(dimethylsiloxane) WT = wild type

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.8b00520. Experimental procedures, protein and DNA sequences, and associated figures (PDF)

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Letter

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Danielle M. Williams: 0000-0001-6763-3775 Chinedum O. Osuji: 0000-0003-0261-3065 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge support from the NSF through the Yale Materials Research Science and Engineering Center (Grant MRSEC DMR-1119826) and from the Raymond and Beverly Sackler Institute for Biological, Physical and Engineering Sciences. C.O.O. acknowledges NSF support under Grant CMMI-1246804 and the facilities of the Yale Institute for Nano and Quantum Engineering (YINQE); L.R. acknowledges support from the NSF (Grant DMR-1307712); D.M.W. acknowledges support from the NIH Cellular and Molecular Biology Training Grant (Grant CMB TG T32GM007223); S.M.P. received funding from a Yale University fellowship; A.E.G. acknowledges funding from the Yale College Dean’s Research Fellowship. E

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DOI: 10.1021/acsanm.8b00520 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX