This work was supported by &#34;Finanziamento di Ricerca di Ateneo&#34; to L. Baccigalupi, project title &#34;SP-LAY: Bacterial spores as live platform for proteins display&#34;.

1. Adsorption reaction
<ol>
	<li>Incubate 2, 5, and 10 &#181;g of mRFP with 2 x 109 of B. subtilis wild-type purified spores in 200 &#181;L of binding buffer, 50 mM sodium citrate, pH 4.0 (16.7 mM sodium citrate dihydrate; 33.3 mM citric acid) for 1 h at 25 &#176;C on a rocking shaker (Figure 1).</li>
	<li>Centrifuge the binding mixtures (13,000 x g for 10 min) to fractionate pellets (P2, P5, and P10) and supernatants (S2, S5, and S10). Store the supernatants for the indirect evaluation of the efficiency of adsorption described in step 3.2.</li>
	<li>Wash the pellets 2x with 200 &#181;L of binding buffer and resuspend them in 100 &#181;L of binding buffer and use it for the next analysis. 
	NOTE: The binding reaction preferentially occurs at a pH value lower than the isoelectric point of the protein; typically, 1.5 M phosphate-buffered saline (PBS), pH 4.0, or 50 mM sodium citrate, pH 4.0, are used.</li>
</ol>
2. Direct evaluation of the efficiency of adsorption
<ol>
	<li>Extraction of surface proteins and western blot analysis
	<ol>
		<li>Take 50 &#181;L of mRFP-adsorbed spores&#39; resuspension (P2, P5, and P10 of step 1.3) and add 50 &#181;L of 2x sodium dodecyl sulfate (SDS)-dithiothreitol (DTT) (0.1 M Tris-HCl, pH 6.8; 2% SDS; 0.1 M DTT) to solubilize surface spore proteins.</li>
		<li>Use the free, purified mRFP and extracts of the same amounts of spores that are not adsorbed to the protein as positive and negative controls, respectively.</li>
		<li>After 45 min of incubation at 65 &#176;C, centrifuge (13,000 x g for 10 min) the mixtures and analyze 10 &#181;L of the extracted proteins (supernatant) by western blot with the monoclonal anti-His antibody recognizing the His tag present at the N-terminal of mRFP (Figure 2A).</li>
	</ol>
	</li>
	<li>Fluorescent microscopy observations 
	NOTE: By using fluorescent heterologous proteins or performing an immunofluorescence analysis, it is possible to localize and quantify the adsorbed molecules by fluorescence microscopy.
	<ol>
		<li>Take 5 &#181;L of the adsorbed spores&#39; resuspension from step 1.3 and add 95 &#181;L of 1x PBS, pH 4.0, to obtain ~1 x 106 spores/&#181;L.</li>
		<li>Place 5 &#181;L of suspension on a microscope slide, cover it with a coverslip previously treated with poly-l-lysine for 30 s, and observe it under a fluorescence microscope.</li>
		<li>For each field, save the phase-contrast microscopy image and fluorescence microscopy image (Figure 2B). 
		NOTE: Alternatively, fluorescent spores can be analyzed by cytofluorimetry. Resuspend a total of 106 spores adsorbed or not-adsorbed with the heterologous protein in 1 mL of 1x PBS, pH 4.0, and analyze the suspension using a flow cytometer (Figure 2C).</li>
	</ol>
	</li>
	<li>Data analysis using ImageJ
	<ol>
		<li>Open the fluorescence microscopy images with ImageJ software (http://rsbweb.nih.gov/ij/) and check to make sure all images are in the 8-bit format (Image | Type | 8-bit).</li>
		<li>Adjust the contrast if necessary (Image | Adjust | Brightness Contrast) and verify that the scale of the image is PIXEL (Image | Set Scale).</li>
		<li>From the Analyze menu, select Set measurements. Make sure to have Area, Integrated Density, and Mean Gray Value selected.</li>
		<li>Draw a line around the spore of interest using any of the drawing/selection tools (i.e., circle, polygon, or freeform) (Figure 3).</li>
		<li>Select Measure from the Analyze menu (or hit cmd + M). A popup box with a stack of values for this first spore appears (Figure 3).</li>
		<li>Repeat these two steps for at least 50 other spores in the field of view chosen to be measured.</li>
		<li>Select several regions without any spores (that have no fluorescence) and repeat the measurement; this will be the background. 
		NOTE: The size is not important. These corresponding background fluorescence values will be used to manually subtract the background.</li>
		<li>Select all the data in the Results window and copy the results into a spreadsheet.</li>
		<li>Calculate the mean of the Integrated Density and Area of the selected spores and of the background fluorescence values and use them to obtain the corrected total-per-cell fluorescence (CTCF) using the formula: CTCF = mean Integrated Density - (mean Area x mean Background fluorescence). 
		NOTE: Alternatively, if all the spores appear well separated, it is possible to analyze all the spores of the image field using the function Analyze Particles, following ImageJ&#39;s instruction. To avoid that the software reads a specific fluorescence or spore aggregates, the dimension of particles has to be set at pixel&#710;2 = 50-200 (Figure 4).</li>
	</ol>
	</li>
</ol>
3. Indirect evaluation of the efficiency of adsorption
<ol>
	<li>Prepare serial dilutions for the purified protein.
	<ol>
		<li>Prepare a first 1.5 mL tube containing 250 &#181;L of purified mRFP at a final concentration of 0.5 ng/&#181;L, using the binding buffer. This volume is sufficient to load two lanes.</li>
		<li>Perform six twofold serial dilutions of 250 &#181;L (final volume) each, using the binding buffer.</li>
	</ol>
	</li>
	<li>Prepare twofold serial dilutions for the supernatant samples containing the unbound mRFP fraction of the adsorption reaction (S2, S5, and S10 from step 1.2).
	<ol>
		<li>Put 100 &#181;L of each supernatant in a 1.5 mL tube and add 100 &#181;L of binding buffer. Perform six twofold serial dilutions of 200 &#181;L (final volume) each, using the binding buffer.</li>
	</ol>
	</li>
	<li>Cut a nitrocellulose membrane (0.45 &#181;m cutoff), 9 cm x 10 cm in size, to cover the area of 5 (number of samples) x 6 (number of dilutions) dots. The membrane should not extend beyond the edge of the gasket of the dot blot apparatus.</li>
	<li>Place the prewet membrane in the dot blot apparatus. Remove any air bubbles trapped between the membrane and the gasket. Cover the unused portion of the apparatus with tape or paraffin film to prevent air from moving through those wells.</li>
	<li>Assemble the dot blot apparatus as described by the manufacturer.</li>
	<li>If a vacuum is used during the assembling, rehydrate the membrane with 100 &#181;L of 1x PBS per well to ensure the uniform binding of the antigen and prevent halos or a weak detection signal.</li>
	<li>Gently remove the buffer from the wells by vacuum. As soon as the buffer solution drains from all the wells, stop the vacuum pump and disconnect it.</li>
	<li>Load the standard in the two most external lanes and the samples in the middle (Figure 5). Fill the appropriate wells with 100 &#181;L of each dilution. The same volume used for each well should ensure a homogeneous filtration of all sample wells.</li>
	<li>Turn on the vacuum pump for 2 min, stop it, and then, allow the sample to filter through the membrane by gravity flow.</li>
	<li>Wash all the wells with 100 &#181;L of 1x PBS and let the vacuum pump run for another 5 min after the washing buffer has been completely drained from the apparatus.</li>
	<li>With the vacuum on, loosen the screws, and carefully open the dot blot apparatus.</li>
	<li>Turn off the vacuum, take the membrane, and process it following a western blot protocol.</li>
	<li>Perform a densitometric analysis of the filter, using appropriate software, such as ImageJ.
	<ol>
		<li>Measure the integrated density of each dot by outlining them with a circle of the same area and using the Analyze/Measure command.</li>
		<li>Make a background correction of the image, drawing a circle in an empty area and measuring its integrated density or using the Process/Subtract Background command.</li>
		<li>Correlate the integrated density of the standard dots with the amount of loaded protein and obtain a calibration line (R2 values for calibration curves should be over 0.95).</li>
		<li>Use the calibration curve to extrapolate the concentration of mRFP of each sample dot.</li>
		<li>Calculate the concentration of the mRFP remaining in the unbound fractions. 
		NOTE: To ensure the correct closing of the dot blot apparatus and, therefore, to subject the membrane to a uniform pressure, tighten the screws by following a diagonally crossed scheme and, then, open the vacuum pump to tighten the screws more strongly.</li>
	</ol>
	</li>
</ol>
4. Spore collection and reuse
<ol>
	<li>For the recycling of the adsorbed spore, perform two adsorption reactions of 2.0 x 109 purified spores with 10 &#181;g of purified GH10-XA xylanase or 10 &#181;g of purified GH3-XT &#946;-xylosidase, as described in steps 1.1-1.3 (Figure 6A).</li>
	<li>Collect the pellets containing the enzyme-adsorbed spores, resuspend them in 50 &#181;L of the optimal buffer for the enzymatic reaction assay (50 mM sodium phosphate buffer at pH 6.5; 2.48689 g/L Na2HPO4, 4.88991 g/L NaH2PO4), and mix them together to obtain a 100 &#181;L mixture of spores adsorbing GH10-XA or GH3-XT.</li>
	<li>Add the substrate (5 mg/mL 4-O-methyl-d-glucuronod-xylan [MGX]) and let the enzyme reactions take place for 16 h at 65 &#176;C.</li>
	<li>Centrifuge the reaction mixture (for 15 min at 13,000 x g) and store the supernatant containing the enzyme reaction product.</li>
	<li>Resuspend the pellet in 100 &#181;L of fresh 50 mM sodium phosphate buffer at pH 6.5 in the presence of a new substrate (MGX) (Figure 6B). 
	NOTE: To adsorb more than one enzyme, it is possible to adsorb both together or one by one independently. The latter possibility facilitates the quantitative analysis of the adsorption efficiency and of the activity of each enzyme, allowing a stoichiometric balance of each enzyme needed for the reactions.</li>
</ol>

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The authors have nothing to disclose.

This spore adsorption protocol is very simple and straightforward. The reaction is strictly dependent on the pH of the reaction buffer and the efficiency of adsorption is optimal at acidic pH values (pH 5.0 or lower). At neutral pH conditions, the efficiency of adsorption is low, and at alkaline pH values, adsorption may not occur. Optimal adsorption is obtained using a volume of 200 &#181;L in 1.5 mL tubes (or keeping a similar ratio) on a rocking shaker.
Adsorption is very tight, and washes with a buffer at the same pH of the reaction buffer do not cause any release of the adsorbed proteins. Washes with alkaline buffers may result in a minimal (generally less than 15%26) release of the adsorbed protein.
The indirect evaluation of the efficiency of adsorption by dot blotting is reliable if several dilutions of purified and unbound protein are analyzed and the densitometric analysis is properly performed. No evidence of degradation of the heterologous proteins has been reported25. The direct evaluation of the efficiency of adsorption greatly depends on the protein that is adsorbed. If the protein is autofluorescent or fluorescently labeled, an ImageJ-assisted analysis provides a quantitative determination of the fluorescence and of the amounts of fluorescent molecules present on the spore. If the protein has an enzymatic activity, a specific enzymatic assay could provide an indication of the amounts of protein present on the spores. However, it is known that the enzymatic activity associated with spores may be increased by a stabilization effect due to the interaction with the spore13. If the adsorbed protein is not fluorescent and does not have an enzymatic activity, the efficiency of adsorption can be evaluated by dot blotting on spores extracted under drastic conditions.
A collection of used spores can be done by a very simple procedure. A washing step with the reaction buffer may be important to remove reaction by-products, while the addition of fresh substrate is essential to initiate a new reaction21.

Display systems are aimed at presenting biologically active molecules on the surface of microorganisms and finding applications in a variety of fields, from industrial to medical and environmental biotechnologies. In addition to phages1,2 and cells of various Gram-negative and -positive species3,4,5,6,7, bacterial spores have also been proposed as display systems by two approaches8,9.
Because of its peculiar structure, namely a dehydrated cytoplasm surrounded by a series of protective layers10, the spore provides several advantages over phage- and cell-based display systems8,9. A first advantage comes from the extreme robustness and stability of spores at conditions that would be deleterious to all other cells10,11. Spore-displayed antigens and enzymes are stable after prolonged storage at room temperature12 and protected from degradation at low pH and high temperatures13. A second advantage of spores is the safety of many spore-forming species. B. subtilis, B. clausii, B. coagulans, and several other species are used worldwide as probiotics and have been on the market for human or animal use for decades14,15. This exceptional safety record is an obvious general requirement for a surface display system and is of particular relevance when the system is intended for human or animal use16. A third, important advantage of a spore-based display system is that it does not have limitations for the size of the molecule that has to be exposed. In phage-based systems, a large heterologous protein may affect the structure of the capsid, while in cell-based systems, it may affect the structure of the membrane or may limit/impair the membrane translocation step17. The protective layers surrounding the spore are composed of more than 70 different proteins10 and are flexible enough to accept large foreign proteins without any evident structural defect or functional impairment8. In addition, with both spore-based display systems, the membrane translocation of the heterologous protein is not required8,9. Indeed, heterologous proteins are either produced in the mother cell cytoplasm and assembled on the spore that is forming in the same cytoplasm or adsorbed on the mature spore8,9.
Spore display was initially obtained by developing a genetic system to engineer the spore surface18. This genetic system was based on i) the construction of a gene fusion between the gene coding for a spore coat protein (used as a carrier) and the gene coding for the protein to be displayed - the presence of the transcriptional and translational signals of the endogenous gene will control the expression of the fusion, and ii) integration of the chimeric gene on the B. subtilis chromosome to grant genetic stability. A variety of antigens and enzymes have been displayed by this recombinant approach, using various spore surface proteins as carriers and aiming at various potential applications, ranging from mucosal vaccine to biocatalyst, biosensor, bioremediation, or bioanalytical tool8,13.
More recently, a different approach of spore display has been developed19. This second system is nonrecombinant and relies on the spontaneous and extremely tight adsorption of molecules on the spore surface9. Antigens19,20 and enzymes13,21 have been efficiently displayed and have revealed that this method is significantly more efficient than the recombinant one. This nonrecombinant approach allows the display of proteins in the native form20 and can also be used with autoclaved, death spores19. The molecular mechanism of adsorption has not been fully clarified yet. The negative charge and the hydrophobicity of the spore have been proposed as properties relevant for the adsorption13,19,22. Recently, it has been shown that a model protein, the red autofluorescent protein (mRFP) of the coral Discosoma, when adsorbed to the spore, was able to infiltrate through the surface layers localizing in the inner coat23. If proved true for other proteins, the internal localization of the heterologous proteins could explain their increased stability when adsorbed to spores23.
In a recent study, two enzymes catalyzing two successive steps of the xylan degradation pathway were independently displayed on spores of B. subtilis and, when incubated together, were able to perform both degradation steps21. Spores collected after the reaction were still active and able to continue the xylan degradation upon the addition of fresh substrate21. Even if a loss of about 15% of the final product was observed in the second reaction21, the reusability of adsorbed enzymes for single, as well as multi-step, reactions is an additional important advantage of the spore display system.
Pan et al.24 reported an additional approach to display heterologous proteins on the spore surface: heterologous proteins (an endoglucanase protein and a beta-galactosidase one) produced in the mother cell during sporulation were spontaneously encased in the forming spore coat, without the need of a carrier. This additional spore display system is a combination of the two approaches described so far. Indeed, it is recombinant since the heterologous proteins were engineered to be expressed in the mother cell during sporulation, while their assembly within the coat was spontaneous and, therefore, nonrecombinant24. However, the efficiency of display of this additional approach remains to be tested and compared with the other two approaches by using the same heterologous proteins.
The present protocol excludes the processes of spore production and purification, which have been described extensively elsewhere24. It includes the adsorption reaction, the evaluation of the efficiency of adsorption by dot-blotting and fluorescence microscopy, and the recycling of adsorbed enzymes for additional reaction rounds.

<table><tbody><tr><td>0.1% Poly-L-lysine solution</td><td>Sigma</td><td>P8920</td><td></td></tr><tr><td>0.45 &amp;micro;m Nitrocellulose Blotting Membrane</td><td>Sartorius</td><td>M_Blotting_Membranes</td><td></td></tr><tr><td>100&amp;times; objective UPlanF1&amp;nbsp;</td><td>Olympus</td><td>microscope equipment</td><td></td></tr><tr><td>Bacillus subtilis&amp;nbsp;strain NCIB3610</td><td>Bacillus Genetic Stock Center</td><td>3A1</td><td></td></tr><tr><td>BD ACCURI C6 PLUS</td><td>BD</td><td>flow cytometer</td><td></td></tr><tr><td>BX51</td><td>Olympus</td><td>Fluorescent microscope</td><td></td></tr><tr><td>Clarity</td><td>Biorad</td><td>1705060</td><td></td></tr><tr><td>DP70 digital camera a</td><td>Olympus</td><td>microscope equipment</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, FITC</td><td>Thermo fisher</td><td>F-2754</td><td></td></tr><tr><td>Goat Anti-Rabbit IgG H&amp;amp;L (HRP)&amp;nbsp;</td><td>Abcam</td><td>ab6721</td><td></td></tr><tr><td>Monoclonal Anti-polyHistidine&amp;minus;Peroxidase antibody</td><td>Sigma</td><td>A7058-1VL</td><td>used to detect adsorbed proteins presenting a 6xhistidine-tag at C- or N- terminal&amp;nbsp;</td></tr><tr><td>SecureSlip&amp;nbsp;glass coverslip</td><td>Sigma</td><td>S1815-1PAK</td><td></td></tr><tr><td>Superwhite Uncharged Microscope Slides</td><td>VWR</td><td>75836-190</td><td></td></tr><tr><td>U-CA Magnification Changer&amp;nbsp;</td><td>Olympus</td><td>microscope equipment</td><td></td></tr></tbody></table>

spore adsorption as a nonrecombinant display system for enzymes and antigens

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure makes the spore extremely stable and resistant and has suggested the use of the spore as a platform to display heterologous molecules. So far, a variety of antigens and enzymes have been displayed on spores of Bacillus subtilis and of a few other species, initially by a recombinant approach and, then, by a simple and efficient nonrecombinant method. The nonrecombinant display system is based on the direct adsorption of heterologous molecules on the spore surface, avoiding the construction of recombinant strains and the release of genetically modified bacteria in the environment. Adsorbed molecules are stabilized and protected by the interaction with spores, which limits the rapid degradation of antigens and the loss of enzyme activity at unfavorable conditions. Once utilized, spore-adsorbed enzymes can be collected easily with a minimal reduction of activity and reused for additional reaction rounds. In this paper is shown how to adsorb model molecules to purified spores of B. subtilis, how to evaluate the efficiency of adsorption, and how to collect used spores to recycle them for new reactions.

Successful adsorption can be assessed by western blotting. Upon the reaction, the mixture is fractionated by centrifugation and washed, and the pellet fraction (Figure 1) is used to extract the surface proteins. The extract is fractionated by SDS polyacrylamide gel electrophoresis (PAGE), electrotransferred to a polyvinylidene fluoride (PVDF) membrane, and reacted against primary and secondary antibodies. The presence of proteins of the expected size, only in the lane loaded with an extract of the adsorbed spores, is indicative of a successful adsorption reaction (Figure 2A).
The efficiency of the adsorption reaction can be evaluated by direct and indirect methods. The direct evaluation of the adsorption efficiency depends on the heterologous protein that has been used and can be performed by fluorescence microscopy (Figure 2B) and cytofluorimetry (Figure 2C) on the pellet fraction after the fractionation of the adsorption reaction. A quantification of the fluorescent signals present on spores can be performed by using the ImageJ software (Figure 3 and Figure 4). An indirect analysis of the adsorption efficiency can be performed by a dot blotting analysis (Figure 5A) of the supernatant fraction containing the unbound protein (Figure 1). A densitometric analysis of the unbound protein (Figure 5B) will then allow scientists to calculate indirectly the amount of protein adsorbed on spores.
Two successive reactions can be catalyzed by a mixture of spores displaying either one of the two specific enzymes (Figure 6A). The adsorbed enzyme(s) can be collected with a simple centrifugation step, washed, and incubated with fresh substrate for a new reaction cycle (Figure 6B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59102/59102fig1.jpg" /> 
Figure 1:General scheme of the adsorption experiment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59102/59102fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59102/59102fig2.jpg" /> 
Figure 2: Direct analysis of the efficiency of the mRFP display. (A) Western blot with mRFP-specific antibody. C+ = free purified mRFP; C- = a protein extract from spores not adsorbed to the protein; P2/P5/P10 = proteins extracted from B. subtilis spores adsorbed with 2, 5, and 10 mg of mRFP, respectively. (B) Immunofluorescence microscopy of adsorbed spores. Immunoreactions were performed with a primary antibody recognizing the adsorbed protein and with a fluorescent secondary antibody conjugated with fluorescein isothiocyanate (FITC). The left panel shows the red intrinsic mRFP fluorescence, the right panel shows the green fluorescence of FITC-conjugated secondary antibody. (C) Flow cytometric analysis of adsorbed spores. The spores reacted with mRFP-specific antibodies and with FITC-conjugated secondary antibodies and, then, were analyzed by cytofluorimetry. The analysis was performed on the entire spore population (10,000 events, ungated). In the left panel, not-adsorbed spores are shown in black, mRFP-adsorbed spores in red. The right panel shows the forward and side scatter (FSC-SSC) dot plot. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59102/59102fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59102/59102fig3.jpg" /> 
Figure 3:&#160;Manual quantification of the fluorescent signal by ImageJ. A fluorescence microscope image of spores, adsorbed with the red fluorescent protein mRFP, analyzed with ImageJ software. A yellow circle has been drawn around one spore to obtain densitometric data (enlarged image). The popup box shows the results of the densitometric analysis of the selected spore. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59102/59102fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59102/59102fig4.jpg" /> 
Figure 4:&#160;Simultaneous quantification of the fluorescent signal by ImageJ. (A) Popup box obtained when selecting Analyze Particle. An interval value of 50-200 pixel^2 has to be set for spores23. (B) Segmentation of the image of Figure 3A after using Analyze Particles (left), and the relative densitometric analysis results (right). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59102/59102fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59102/59102fig5.jpg" /> 
Figure 5:&#160;Dot blotting and densitometric analysis. (A) Dot blotting with serial dilutions of the purified mRFP in duplicate (Std1 and Std2) and the supernatant (S10, S5, and S2) of the adsorption reaction performed with 10, 5, and 2 &#181;g of mRFP, respectively23. (B) The dot blotting of panel A is used for the densitometric analysis. The circles indicate the area used to quantitate the density of the signals. The panel on the right reports an example of results obtained with the densitometric analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59102/59102fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59102/59102fig6.jpg" /> 
Figure 6:&#160;Conversion of xylan by reusable spores. (A) General scheme of xylan degradation. (B) Spores displaying the xylanase or the &#946;-xylosidase enzymes, when mixed together, catalyze the two-step degradation of xylan. After the reaction, the sample is fractionated by centrifugation. The supernatant contains the reaction product, while the pellet contains spore-bound enzymes that can be reused by adding fresh substrate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59102/59102fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens at JoVE.com

Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

This protocol focuses on the use of bacterial spores as a &#34;live&#34; nanobiotechnological tool to adsorb heterologous molecules with various biological activities. The methods to measure the efficiency of adsorption are also shown.

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure ...

spore-adsorption-as-nonrecombinant-display-system-for-enzymes

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

6.6K Views.  Federico II University. Levende bacteriële sporen met antigeen of enzymen zijn functioneel actieve nanodeeltjes die kunnen worden gebruikt als mucosale vaccins of biokatalysten. In principe kan elk eiwit efficiënt worden geadsorbeerd op sporen. Deze techniek is eenvoudig en vereist geen genetische manipulatie.Daarom is er geen sprake van het vrijkomen van recombinante organismen in het milieu. Naast mucosale vaccinologie voor mens en dier kunnen sporen met enzymen worden ontwikkeld als herbruikbare biokatal...

Video: Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

Biochemistry

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins with high specificity but low affinity. Through phage display, ubiquitin variants (UbVs) can be engineered such that they exhibit improved affinity over wildtype ubiquitin and maintain binding specificity to target proteins. Phage display utilizes a phagemid library, whereby the pIII coat protein of a filamentous M13 bacteriophage (chosen because it is displayed externally on the phage surface) is fused with UbVs. Specific residues of human wildtype ubiquitin are soft and randomized (i.e., there is a bias towards to native wildtype sequence) to generate UbVs so that deleterious changes in protein conformation are avoided while introducing the diversity necessary for promoting novel interactions with the target protein. During the phage display process, these UbVs are expressed and displayed on phage coat proteins and panned against a protein of interest. UbVs that exhibit favorable binding interactions with the target protein are retained, whereas poor binders are washed away and removed from the library pool. The retained UbVs, which are attached to the phage particle containing the UbV's corresponding phagemid, are eluted, amplified, and concentrated so that they can be panned against the same target protein in another round of phage display. Typically, up to five rounds of phage display are performed, during which a strong selection pressure is imposed against UbVs that bind weakly and/or promiscuously so that those with higher affinities are concentrated and enriched. Ultimately, UbVs that demonstrate higher specificity and/or affinity for the target protein than their wildtype counterparts are isolated and can be characterized through further experiments.

Ubiquitination is a critical protein post-translational modification, dysregulation of which has been implicated in numerous human diseases. This protocol details how phage display can be utilized to isolate novel ubiquitin variants that can bind and modulate the activity of E3 ligases that control the specificity, efficiency, and patterns of ubiquitination.

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing need for the development of methodologies that improve the detection of antigens immobilized on the surface of spores together with their quantification. Flow cytometry-based analyses have been previously proposed as fast, reliable, and specific approaches for detecting labeled cells of B. subtilis. Herein, we propose the use of flow cytometry to evaluate the display efficiency of a fluorescent antibody (FA) on the surface of the spore and quantify the number of spores using counting beads. 
For this, we used ethidium bromide as a DNA marker and an allophycocyanin (APC)-labeled antibody, which was coupled to the spores, as a surface marker. The quantification of spores was performed using counting beads since this technique demonstrates high accuracy in the detection of cells. The labeled spores were analyzed using a flow cytometer, which confirmed the coupling. As a result, it was demonstrated that DNA labeling improved the accuracy of quantification by flow cytometry, for the detection of germinated spores. It was observed that ethidium bromide was not able to label dormant spores; however, this technique provides a more precise determination of the number of spores with fluorescent protein coupled to their surface, thus helping in the development of studies that focus on the use of spores as a biotechnological platform in different applications.

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

This protocol focuses on the use of flow cytometry and counting beads to quantify bacterial spores labeled with ethidium bromide. The method is also efficient for analyzing the covalent coupling of proteins on the surface of intact spores.

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing ...

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic biotinylation by the E. coli biotin-protein ligase BirA is highly specific and allows for the biotinylation of target proteins in their native environment; however, the current usage of BirA mediated biotinylation requires the presence of a synthetic acceptor peptide (AP) in the target protein. Therefore, its application is limited to proteins that have been engineered to contain the AP. The purpose of the present protocol is to use the bacterial display of a peptide derived from an unmodified target protein to select for BirA variants that biotinylates the peptide. The system is based on a single plasmid that allows for the co-expression of BirA variants along with a scaffold for the peptide display on the bacterial surface. The protocol describes a detailed procedure for the incorporation of the target peptide into the display scaffold, creation of the BirA library, selection of active BirA variants and initial characterization of the isolated BirA variants. The method provides a highly effective selection system for the isolation of novel BirA variants that can be used for the further directed evolution of biotin-protein ligases that biotinylate a native protein in complex solutions.

Here we present a method to select for novel variants of the E. coli biotin-protein ligase BirA that biotinylates a specific target peptide. The protocol describes the construction of a plasmid for the bacterial display of the target peptide, generation of a BirA library, selection and characterization of BirA variants.

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic ...

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

Engineering Antiviral Agents via Surface Plasmon Resonance

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...

Bioengineering

A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain

Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane vesicles (OMVs) are mainly secreted by gram-negative bacteria during average growth, with a nano-sized diameter and self-adjuvant activity, which may be ideal for vaccine delivery. OMVs have functioned as a multifaceted delivery system for proteins, nucleic acids, and small molecules. To take full advantage of the biological characteristics of OMVs, bioengineered Escherichia coli-derived&#160;OMVs were utilized as a carrier and SARS-CoV-2 receptor-binding domain (RBD) as an antigen to construct a &#34;Plug-and-Display&#34; vaccine platform. The SpyCatcher (SC) and SpyTag (ST) domains in Streptococcus pyogenes were applied to conjugate OMVs and RBD. The Cytolysin A (ClyA) gene was translated with the SC gene as a fusion protein after plasmid transfection, leaving a reactive site on the surface of the OMVs. After mixing RBD-ST in a conventional buffer system overnight, covalent binding was formed between the OMVs and RBD. Thus, a multivalent-displaying OMV vaccine was achieved. By replacing with diverse antigens, the OMVs vaccine platform can efficiently display a variety of heterogeneous antigens, thereby potentially rapidly preventing infectious disease epidemics. This protocol describes a precise method for constructing the OMV vaccine platform, including production, purification, bioconjugation, and characterization.

The present protocol describes the bioengineering of outer membrane vesicles to be a "Plug-and-Display" vaccine platform, including production, purification, bioconjugation, and characterization.

Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

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