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Reporter Immobilization Assay (REIA) for Bioconjugating Reactions Martin Schatte,*,†,‡ Marco Bocola,† Teresa Roth,‡ Ronny Martinez,† Erhard Kopetzki,§ Ulrich Schwaneberg,*,† and Mara Bönitz-Dulat*,‡ †

Lehrstuhl für Biotechnologie, RWTH Aachen University, 52074 Aachen, Germany Enzymes and Protein Technologies and §Large Molecule Research, Roche Diagnostics GmbH, 82372 Penzberg, Germany

‡

S Supporting Information *

ABSTRACT: Enzymes able to ligate biomolecules are emerging tools to generate site-specific bioconjugates. In this study we present a detection and screening method for bioconjugating enzymes which overcomes limitations of analytical methods such as HPLC or MS. These techniques are experimentally demanding and often limited in sensitivity and throughput compared to enzymatic assays. The principle of this Reporter Immobilization Assay (REIA) is the ligation of a reporter enzyme to a peptide carrying an affinity handle, which can be utilized for its isolation. The REIA system exhibits a high sensitivity with a linear range down to 1 μg/mL (55 nM), a variation coefficient of 6.5%, and can be performed cost-efficiently in 96-well microtiter plate format. The application of this assay allowed the characterization of a thiol transpeptidase sortase from S. aureus which is an important drug target and a biotechnological tool for ligation and modification of proteins. Thereby, yet-undetectable promiscuous activity of sortase could be detected, e.g., the acceptance of alanine as nucleophile. In addition, we were able to provide evidence that the REIA is suitable for high throughput screening of enzyme libraries using crude cellular extract with a throughput of 600 samples per hour.

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be screened to find the appropriate enzymes for specific applications. For bioconjugating enzymes, however, there is stillto our knowledgeno sensitive high throughput assay for product formation described. So far, product formation is monitored via mass spectrometry (MS) or high performance liquid chromatography (HPLC) to determine weight or hydrophobicity changes between educts and products. These methods, though suitable, are complex in experimental handling, time-consuming, costly, and difficult to perform in the context of mutant library screening. For peptide ligating enzymes like transglutaminases and sortases, which show a Ping-Pong mechanism (Figure 1B), detection systems for the acylation step were developed.10,11 Sortases have emerged in the field of bioconjugation since they were first described in 199412 with more than 100 publications in 2014. Sortases are an important enzyme family, not only as a biotechnological tool for protein modification and ligation, but also as a potential new antibiotic drug target.13 This makes sortases the most widely used bioconjugating enzymes and is therefore a suitable example to evaluate assays for this class of enzymes. Sortases are transpeptidases which specifically recognize a Sort-tag motif (e.g., LPXTG for Sa-SrtA, X = any amino acid) and perform a C-terminal cleavage of the peptide bond between threonine and glycine.11,14 The C-

INTRODUCTION Bioconjugation is an attractive and powerful method for the mild and site-specific modification of proteins.1 Among other applications, of highest impact are enzyme- and fluorescentcoupled immunoassays,2 cellular imaging,3 creation of posttranslational modifications,2 and the production of antibody− drug conjugates.4 Most techniques for the generation of bioconjugates, such as using N-hydroxysuccinimide ester, maleimides, or iodocetamide, are rather unspecific,5 since they rely on the accessibility and reactivity of the corresponding amino acids which can lead to unwanted side effects.2 Therefore, site-specific conjugation tools are an important technology for future bioconjugating approaches. Due to their high degree of stereo-, regio-, and chemoselectivity, enzymes are promising candidates for selective catalysis of this type of reaction. Different enzymes have been applied for specific bioconjugation, the most prominent ones being glycosyltransferases, transglutaminases, and transpeptidases like sortase A from Staphylococcus aureus (Sa-SrtA).6 However, for many applications such as industrial bioconjugate production or approaches where the existing recognition motives is obstructive, no suitable bioconjugating enzyme is available.6 To overcome these limitations, improved variants generated through protein engineering using, e.g., directed evolution,7 can be generated. This was shown by Chen et al. for an affinity improved sortase variant.8,9 In directed evolution campaigns, the prerequisite is a sensitive and reliable high-throughput screening system, since large libraries have to © XXXX American Chemical Society

Received: February 25, 2016 Revised: May 12, 2016

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DOI: 10.1021/acs.bioconjchem.6b00111 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

specificity for N-terminal glycine residues. So far no Sa-SrtA activity for other N-terminal residues than glycine could be detected.15 The fluorogenic sortase assay reported in the literature19 uses an Abz-LPETG-Dnp-NH2 substrate (Figure 1A). The fluorescent Abz (ortho-aminobenzoyl) dye is intramolecularly quenched by the Dnp (2,4-dinitrophenyl).11 After the formation of the acyl-enzyme intermediate and the release of the G-Dnp, the quenching of Abz is abrogated and an increase in fluorescence signal can be observed. As a limitation of this assay it has been shown that the increase in fluorescence intensity upon educt cleavage is not linear due to inner filter effects at higher substrate concentrations.19 Further on, the measured signal is independent of ligation or hydrolysis of the bound intermediate enzyme−peptide complex.19 Therefore, the major drawback of these educt-based transglutaminase and sortase assays is that only the reaction of the enzyme with the first substrate can be monitored irrespective of the following reaction. Hydrolysis leads to reduced product formation due to the generation of a free carboxylate end group which is an undesired side product. Thus, there is a need for an efficient and sensitive product-based assay for the detection of complete bioconjugation reactions. Protein engineering approaches of enzymes are mainly limited by the number of mutants or parameters which can be screened in a certain time.20 A high throughput screening (HTS) assay that can monitor both steps, the generation of the enzyme-intermediate and the ligation will offer many advantages. In this work we developed a product-based bioconjugation assay, employing Sa-SrtA as model enzyme. With the presented REIA it was possible to perform a detailed characterization of the substrate specificity of Sa-SrtA. In addition, Sa-SrtA and a recently reported variant9 were analyzed regarding their altered properties directly from crude cellular extract using the REIA screening system in 96-well MTP format.

Figure 1. (A) Sortase assay published by Ton-That et al. uses an Abz fluorophore, initially quenched by Dnp and linked with a LPXTG peptide. The nucleophilic attack of the sortase cleaves the peptide and leads to abrogation of the Abz quenching. No further step can be monitored. (B) Sortases exhibit a Ping-Pong mechanism. During the first reaction step the sortase attacks the LPXTG motive by cleaving the bond between T and G, thereby destroying the FRET between Cy5 and FITC. In a second step the formed enzyme intermediate gets attacked by a glycine (G), linked to a Black Hole Quencher 2 (BHQ), forming a new peptide bond. In the ligation product the linked BHQ2 should quenches the fluorescence of Cy5. (C) Reporter Enzyme Immobilization Assay (REIA). The reporter enzyme (GDH) carries the first substrate (LPXTG1) at the c-terminus and the affinity handle (biotin) carries the second substrate (glycine). After the reaction with the bioconjugating enzyme of interest (SrtA) the ligation product (LPXTG2) is formed and the reporter is fused to the affinity handle. The reaction mixture is transferred to a streptavidin matrix (SA), which can be microtiter plates (MTP) or magnetic beads (MAG) where the biotin is immobilized and not ligated reporter enzyme is washed away. In the last step the activity of the immobilized reporter enzyme is measured.

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RESULTS AND DISCUSSION Ideally, detection as well as screening systems for bioconjugating reactions should monitor the complete ligation reaction, not only an intermediate state. Likewise, an analytical system to calculate unknown enzyme concentration must be reproducible with a low variation between measurements and show a broad linear range between readout and enzyme concentration. To detect promiscuous activity, a high sensitivity of the system is needed. In contrast, for screening systems, the key parameter is the achieved throughput. Another big advantage for enzyme screening systems is the possibility to screen in crude cellular extract, allowing quick assessment of every single variant without the need of purification. As explained above, current systems for monitoring bioconjugating reactions still have room for improvement, which we have addressed in the development of the FRET and REIA system described here. FRET Based Assay. As an improvement to the standard approach of determining only the cleavage rate of an internally quenched peptide, we investigated a detection system for the cleavage and the formation of a bioconjugation product. To achieve this objective, a FRET-based assay between the target peptides was proposed (Figure 1B).21 Initially, a LPETG peptide (Cy5-ULPETGGG-FITC) which contained an Nterminal Indodicarbocyanine (Cy5) and a C-terminal Fluorescein isothiocyanate (FITC) was synthesized, where the energy transfer from the FITC to the Cy5 should be prevented upon cleavage of the peptide. In a second step, a peptide

terminal fragment of the Sort-tag is released while the Nterminal part stays covalently bound to the sortase via a thioester bond to threonine. This acyl-enzyme intermediate is then released by a nucleophile attack of the N-terminus of a polyglycine in a transpetidation reaction or by a water molecule in a hydrolysis reaction.14,15 Sortases can be grouped in six families, depending mainly on the physiological function described in their host organism.16 These functions include immobilization of proteins in the cell wall,12 spore formation,17 and polymerization of pili subunits.18 Family A sortases exhibit mainly three different specificities (glycine, alanine, and diaminopimelic acid) depending on the kind of cell wall crossbridge prevalent in the host peptidoglycan.16,17 Sortase A from Staphylococcus aureus (Sa-SrtA) is the most applied and consequently the best characterized sortase. An approach to improve its affinity to the Sort-tag, via yeast display, has been described.9 In S. aureus the natural nucleophile has been identified as pentagylcine.12 Accordingly, this sortase exhibits B


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cytometers include lasers which can activate Cy5 and FITC (488 nm, blue laser) but not Abz (355 nm, UV laser). However, quenching of Cy5 was not detectable (SI, Figure S1). Due to the high Km of Sa-SrtA (5.5 mM for LPXTG),19 high concentrations of the substrate are required. This facilitates the intermolecular self-quenching of the fluorophores and the formation of hydrophobicity-induced aggregates. Consequently, we tested different fluorophore−quencher combinations. The constructs are shown in SI, Table S1. Unfortunately, none of the combinations provided an efficient system to monitor the product formation of the ligation reaction on a product base. Reporter Immobilization Assay (REIA). Most bioconjugation applications are not restricted to peptides, but include antibodies, enzymes, or DNA as targets. Furthermore, it was reported that the properties of the functionalization of the two substrates can influence their ligation.2,24 Commonly used assay systems employ small, labeled peptides as substrates for either continuous (fluorescence quenching or FRET) or discontinuous (HPLC) activity measurement, restricting the number of operable experiments. To overcome these limitations, we developed a strategy for the differentiation of the product of a bioconjugation reaction from the educts and their quantitative detection. The method relies on the attachment of an affinity handle to an easily detectable reporter. Via the affinity handle, the successfully linked reporter protein can be immobilized and separated from the residual free reporter (Figure 1C). According to the principle, we named it REIA. In this study, a biotinylated GGG-peptide was used as affinity handle and a recombinant, soluble quinoprotein glucose dehydrogenase (GDH) from A. calcoaceticus harboring a C-terminal recognition sequence was used as reporter. An important aspect of this assay is the load capacity of the immobilization surface as well as the ability to efficiently wash the surface. Different streptavidin coated surfaces were used to bind the biotinylated reporter, being microtiter plate (MTP) and magnetic beads (MAG). Both types of surfaces possess specific advantages. High throughput screenings may be performed with a MTP system, which can be washed automatically. However, their binding capacity is limited to approximately 20 nmol of biotin per well. This is not sufficient

nucleophile containing a C-terminal Black Hole Quencher 2 (BHQ2) was applied as nucleophile, which should suppress the fluorescence of the Cy5 after ligation. The objective of the designed peptides, shown in Figure 1, was the monitoring of the ligation reaction via quenching of Cy5 fluorescence (excitation: 650 nm, emission: 670 nm). Additionally, the cleavage of the LPETG motif should result in increased FITC fluorescence (excitation: 480 nm; emission: 530 nm) and decrease the FRET to Cy5, measured by 480 nm excitation and 670 nm emission. Similar to the approach reported by Ton-That and co-workers,11 we were able to monitor the acylation reaction, namely, the cleavage of the Cy5ULPKTGGGRRC-FITC. This was indicated by the increase of fluorescence of FITC. (Figure 2).

Figure 2. Emission spectra (excitation: 480 nm) of the sortase reaction mixture ca. 50 μM Cy5-LPETGGGRRC-FITC, ca. 50 μM GGGWWBHQ2 and 10 μM Sa-SrtA, recorded at the indicated time points, color coded from red (0 min) to blue (420 min).

Up to now, the fluorophores used (Cy5 and FITC) were not shown to be suitable for the detection of sortase activity. The expansion of the spectrum of useful fluorophores shown in this work gives rise to new applications where the previously described fluorophores could not be used; e.g., most flow

Figure 3. (A) Sortase catalyzed biotinylation of GDH. Reporter enzyme activities for very low Sa-SrtA concentrations (1−10 μg/mL (50−500 nM)) after 2 h incubation under standard sortase reaction conditions (20 μM GDH-LPKTG. 20 μM tetra glycine-biotin in 50 mM Tris-HCL buffer pH 7.5. 200 mM NaCl. 10 mM CaCl) are displayed. (B) Sortase catalyzed biotinylation of s-GHD using alanine as nucleophile. Reporter enzyme activities for Sa-SrtA concentrations between 0.1 and 1 mg/mL (5.5−55 μM) after 2 h incubation under standard sortase reaction conditions (180 μM GDH-LPKTG. 180 μM tetra alanine-biotin in 50 mM Tris-HCL buffer pH 7.5. 200 mM NaCl. 10 mM CaCl2) are displayed. In both graphs standard deviations of three independent measurements are shown in the error bars. Activity of reporter enzyme is displayed in dE/min. C


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Figure 4. (A) Sortase catalyzed biotinylation of GDH using low substrate concentrations (2 μM). Reporter enzyme activities were analyzed using different Sa-SrtA concentrations (5−50 μg/mL (0.25−2.5 μM)) along with the lower concentration of biotinylated substrate; standard REIA conditions were applied (2 μM tetra glycine-biotin, 20 μM GDH-LPKTG. in 50 mM Tris-HCL buffer pH 7.5. 200 mM NaCl. 10 mM CaCl). (B) Sortase catalyzed biotinylation of GDH using high substrate concentrations (200 μM). Reporter enzyme activities were analyzed using different SaSrtA concentrations (10−50 μg/mL (0.5−2.5 μM)) along with the higher concentration of biotinylated substrate; standard REIA conditions were applied (200 μM tetra glycine-biotin, 20 μM GDH-LPKTG. in 50 mM Tris-HCL buffer pH 7.5. 200 mM NaCl. 10 mM CaCl). In both graphs standard deviations of three independent measurements are shown in the error bars. Activity of reporter enzyme is displayed in dE/min.

before due to lack of sensitivity, but clearly shown using the developed REIA assay system. For analytical usage of the REIA system, SA-MAG was used in a concentration in which streptavidin exceeds the concentration of biotin at least by a factor of 3. This is critical when using different amounts of the biotinylated substrate. To prove that the REIA is suitable for a wide range of substrate concentrations, measurements at low (2 μM) and high (200 μM) substrate concentrations were performed. Therefore, slight changes in the REIA protocol were implemented. When using 2 μM biotinylated substrate, the reaction mixture was only diluted by a factor of 5. When using 200 μM biotinylated substrate, the reaction mixture was diluted by a factor of 50 and doubled concentration of SA-MAGs was used (Figure 4). In both cases the streptavidin still exceeds the biotin concentration and a linear correlation between reporter enzyme activity and sortase concentration could be shown. However, at 200 μM biotinylated substrate and higher Sa-SrtA concentrations the limit of the assay starts to show up, indicated by several outliers. However, for concentrations in the 2−20 μM range, a linear correlation with low standard deviations could be shown. The reliability of REIA over two magnitudes of substrate concentrations renders the assay generally applicable for multiple reaction conditions. High Throughput Screening of Bioconjugating Enzymes. SA-MTPs are used for screening. In this manuscript, when SA-MTP are used the concentration of biotin exceeds the concentration of streptavidin, different from the analysis using SA-MAG. A prerequisite for a high throughput assay used in protein engineering is the ability to screen mutant libraries in nonpure solutions containing the enzyme of interest. This requires a robust and specific assay since crude extracts contain only limited quantities of the target enzyme and a high amount of promiscuous activities. Thus, the robustness of REIA was challenged by substrate affinity binding assay in the MTP scale. Transpeptidation assays applied in high throughput studies commonly monitor the substrate cleavage at high substrate and enzyme concentration, thus neglecting hydrolysis and final product formation.23 Screening for enzymes with lower KM

for analytics of bioconjugating reactions but can be overcome by the use of magnetic beads facilitating the application of higher quantities of streptavidin and exhibiting less background signal (SI, Table S2). REIA as Reliable Analytical Assay. SA-MAG are used for analysis. In this manuscript SA-MAG are always used in a concentration in which the streptavidin exceeds the concentration of biotin at least by a factor of 3. Analytical assays must be sufficiently sensitive and reproducible with a minimized variance.20 Sortase assays reported in the literature use Sa-SrtA concentrations in a 10−200 μg/mL (500−10 000 nM) range.11,19 However, most enzyme assays work with enzyme concentrations in the low nanomolar rage.22 To evaluate the sensitivity of the REIA, concentrations of 1−10 μg/mL (55−550 nM) Sa-SrtA were mixed with 20 μM of each substrate and incubated for 2 h. This mixture was transferred to streptavidin coated magnetic beads. After washing, the activity of the reporter enzyme was detected. In Figure 3A it can be seen that at 1−10 μg/mL (55−550 nM) Sa-SrtA enzyme concentrations, a linear correlation between sortase concentration and reporter enzyme activity could be observed. Therefore, REIA is a very sensitive analytical tool for monitoring sortase activity and examining the reaction conditions. Compared to HPLC analysis, where more than 10 μg/mL sortase is used, the REIA exhibits a 15-fold higher sensitivity.19 Additionally, this sensitivity level was achieved with a very low coefficient of variation (average 6.5%). Previous studies tried to find alternative nucleophiles than glycine, e.g., alanine, to characterize the nucleophile pocket, but failed due to low sensitivity of the assay used.15 To investigate if we can find promiscuous activity of Sa-SrtA, we used the REIA with alanine instead of glycine as nucleophile. Enzyme concentrations from 0.1 to 1 mg/mL (5.5−55 μM) were analyzed for their activity with alanine (Figure 3B). With all tested sortase concentrations ligation product could be detected. A linear correlation between applied sortase concentration and obtained product concentration is given. This promiscuous activity of Sa-SrtA with alanine as nucleophile could not be shown with assays used in the studies D


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exhibit a low standard deviation. However, to obtain quantitative results with higher concentrations of the affinityhandle-substrate, the capacity of the capturing matrix and the amount of the reaction mixture analyzed have to be carefully adjusted. Compared to the photometrical assay, presented by Ton-That et al., the REIA recognizes the true ligation product of a bioconjugation reaction, independent of hydrolyses. This is especially important because most engineered sortases have a dramatically fast hydrolysis rate.26,27 The very accurate activity measurement (6.5% coefficient of variation) down to 1 μg/mL (55 nM) Sa-SrtA is outstanding compared to the sortase assays reported up to now. This was shown with the newly found promiscuous activity of Sa-SrtA to alanine as nucleophile substrate. The throughput of over 600 samples per hour enables the screening of large mutant libraries, which is extremely important for direct evolution campaigns. Additionally, with the use of a RAQ it is even possible to screen these libraries for different parameters, e.g., affinity from crude cellular lysate. Analyzing Additional Bioconjugation Reactions. To show that the REIA is not limited to sortase catalyzed reaction, we tried to analyze the microbial transglutaminase (mTG), another frequently used bioconjugation enzyme. This enzyme class catalyzes the reaction of a glutamine residue with a primary amine, mainly lysine, thereby releasing ammonium and forming an iso-peptide bond between the two substrates.24 Due to the high promiscuity of mTG25 concerning the amino acids flanking the glutamine residue it was possible to use the reporter enzyme (GDH) without any additional high affinity recognition tag. mTG concentrations of 1−6 μg/mL were analyzed using the wt-GDH and a lysine-biotin construct and incubated for 1 h. All other assay parameters were used as described for the REIA analyzing sortase reaction. The result is shown in Figure 5C; a linear correlation between the mTG concentrations and the measured reporter GDH activity was detected. The mTG concentration range as well as the performance of the assay is similar to the sortase setup. However, mTG shows an up to 100-fold higher catalytic efficiency than Sa-SrtA, meaning that even lower concentrations could be detected by adding a high affinity tag for mTGs, to the reporter GDH. To show the general applicability of the REIA, analysis to monitor nonenzymatic bioconjugation reactions was performed. The most widely, but unspecifically, used method is the reaction of an activated N-hydroxysuccinimiede ester with a primary amine.26 Similar to the mTG system, no additional recognition tag on the reporter enzyme (GDH) was needed. An NHS ester liked to biotin was used as affinity handle. Two future adjustments had to be done: one was the use of HEPES instead of the amine containing TRIS buffer, and instead of using IAA to stop the reaction 100 mM lysine was used to quench the reaction. A linear correlation between NHS ester concentration and the analyzed reporter GDH activity could be detected. This finding supports the idea that the REIA is generally applicable to a wide range of bioconjugating reactions. Adopting the REIA to Fluorophores as Reporter. A future application of the REIA is the use of fluorescent labels as reporters. To address this question the reporter enzyme GDH was replaced with the fluorogenic substrate LCR640ULETGGGRR-OH. The reaction conditions are kept as described for the sortase assay and an SA-MTP with low intrinsic fluorescence was used (Figure 6).

value is important for different biotechnical applications such as in clinical chemistry where complete conversion is essential.22 This represents a specific challenge for high throughput methods. Here we used the REIA for product based Sa-SrtA activity screening in crude cell extract, at high and low substrate concentrations, to enable a fast and robust screening for bioconjugation enzymes like Sa-SrtA. In order to approximate the binding affinity of an enzyme in a screening assay, one possibility is to calculate the quotient of the enzymatic activity at two different substrate concentrations,26,27 chosen in the KM-concentration range of the enzyme. The calculated value is defined as Relative Affinity Quotient (RAQ) (SI, Figure S2). RAQ =

activity of reporter[low substrate] activity of reporter[high substrate]

An enzyme variant with improved affinity can be identified via higher activity quotients compared to the wild type enzyme. To prove that the REIA can achieve this objective, the P94S variant of Sa-SrtA, recently published by Chen et al.,9 were analyzed. This variant exhibits an improved KM-value for the LPXTG substrate. For the determination of the RAQSort‑tag value, sortase activities were measured at substrate concentrations of 20 μM and 4 μM. For the wild type enzyme, a RAQ of 0.249 ± 0.024 was measured in 16 independently grown and lysed wild type cultures (SI, Table S3). For the P94S variant of Sa-SrtA, with 0.346 ± 0.016 (SI, Table S3) a significantly higher RAQ could be detected. In Table 1 the RAQ-values of Sa-SrtA Δ59 WT Table 1. RAQSort‑tag of Sa-SrtA Δ59 WT and P94S Mutanta sortase variant

Km Sort‑tag9

RAQSort‑tag

Sa-SrtA Δ59 WT Sa-SrtA Δ59 P94S9

7.6 mM ± 0.6 2.5 mM ± 0.5

0.241 ± 0.025 0.349 ± 0.015

a

The RAQSort‑tag of mutant and wild type were calculated by the activities with different substrate concentrations (20 and 4 μM) and evaluated with a t-value of 5 × 10−14 difference. The reference Kmvalues shown here where published by Chen et al. using HPLC detection as reference.8

were compared with the variant Sa-SrtA Δ59 P94S exhibiting a lower Km (Sort-tag) value. The change in RAQSort‑tag value determined in crude enzyme extract corresponds to the reported Km-values. Thus, the matching values, as well as the low standard deviations, prove the feasibility of the REIA for detection of highly affine variants from crude extract. With the described setup, about 600 measurements per hour can be performed. As described in the Materials and Methods section in detail, streptavidin coated MTPs where used for the REIA-screening approach. However, the immobilization capacity of the plates is a limiting factor in determining the absolute concentration of immobilized product. The biotin used as nucleophile substrate in the assay exceeds the amount of streptavidin on the plate by a factor of approximately 3. Accordingly, the ratio between biotinylated enzyme and biotinylated substrate was analyzed. To determine the appropriate conditions for a reliable activity detection of the biotinylated product, the time for reaching the equilibrium of the binding process was measured (SI, Figure S3). After 25 min, a plateau is reached indicating that the equilibrium is established. Consequently, after this time point the immobilization process is completed and measurements E


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Figure 5. (A) Transglutaminase reaction schema. Microbial transglutaminase (mTG) attacks a glutamine residue in the reporter enzyme (GDH) and releases ammonium by the formation of an acyl intermediate which gets resolved by the attack of amine, in this case linked to biotin. The reaction establishes an iso-peptide bond between the GDH and the biotin. (B) Activated N-hydroxysuccinimide ester (NHS-ester) reaction. In this case the biotin is linked to an NHS-ester which gets attacked by an amine of a lysine residue from the GDH. The reaction establishes an iso-peptide bond between the GDH and the biotin. (C) Standard REIA reaction conditions (20 μM lysine-Biotin, 20 μM GDH, in 50 mM Tris-HCL buffer pH 7.5, 200 mM NaCl, 10 mM CaCl2 for 1 h) were applied to 1−6 μg/mL mTG and the resulting GDH activities are shown. (D) 2−20 μM NHS-esterbiotin were suspected to adopted REIA reaction conditions (20 μM GDH, in 50 mM Hepes buffer pH 7.8, 200 mM NaCl, 10 mM CaCl for 1 h). The resulting GDH activities are shown. In both graphs standard deviations of three independent measurements are shown in the error bars. Activity of reporter enzyme is displayed in dE/min.

analyzed. Therefore, the developed REIA enables the analysis of kinetic parameters and substrate spectra of different reactions and can generate new insights in substrate recognition and catalytic mechanism of bioconjugating enzymes, such as for the alanine acceptance of Sa-SrtA (Figure 3B). With the activity analysis of a second bioconjugation enzyme, the microbial Transglutaminase, and the determination of the uncatalyzed N-hydroxysuccinimide ester reaction, the broad applicability of REIA was shown. Systems like mTG of NHS esters are consider to be promiscuous (both attack amines mainly independent from surrounding residues), which can lead to unwanted side effects if using a complex enzyme as reporter. In such cases the REIA can be performed using fluorophores linked to small peptide substrates to reduce unwanted side effects.The robustness and the throughput of over 600 samples from crude cellular lysate per hour makes the REIA a valuable HTS tool in the search for new bioconjugating enzyme variants. The application of the REIA system is not limited to bioconjugate chemistry; for instance, it can be extended to screen inhibitors of clinically relevant bioconjugating enzymes, such as sortases as new antibiotic drug targets27 or transglutaminases which are key players in the development of celiac disease.28

A linear correlation between Sa-SrtA concentration and the fluorescence intensity could be observed, indicating that REIA can be performed with fluorophores as reporters as well. Why it is possible to monitor the ligation reaction, with the same fluorogenic substrate, in the REIA system but not in the FRET based assay, can have multiple causes. The LCR640 was especially designed to be more water-soluble than other fluorophores, like Cy5. Additionally only one hydrophobic fluorophore species is present in the reaction mixture and only a portion of this will be transferred to the SA-MTP. These changes in the REIA system compared to the FRET assay should reduce the formation of aggregates during the reaction as well as the self-quenching during the analysis.

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CONCLUSIONS REIA is a highly reproducible detection system in 96-well-plate format with an excellent coefficient of variation of 6.5% and a linear rage of 1−10 μg/mL Sa-SrtA. The improved sensitivity using magnetic beads was shown by the observation of alanine acceptance of Sa-SrtA, which is not detectable by current standard methods. Additionally, REIA enables one to monitor product formation, not only the generation of the thioester intermediate. Through different dilutions of the terminated reaction mixture and consequent use of different amounts of SA-MAG, a broad range of substrate concentrations can be F


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Glucose Dehydrogenase (GDH). The gene of the soluble PQQ-dependent glucose dehydrogenase from Acinetobacter calcoaceticus (EC 1.1.5.2, GDH)) used by Duine and coworkers29 with or without a C-terminal addition of a SGALPKTGGSGS sequence was ordered from Geneart/ Regensburg and cloned via EcoR1 and HindIII into pkk177 (constitutive promoter) and transformed in Escherichia coli XL1 blue cells. The cells were cultivated (overnight, 5 L shaking flasks filled with 2 L LB-media, 37 °C, 140 rpm) and harvested via centrifugation. The cells were homogenized via French press (1 kbar). The extract was centrifuged and the supernatant further processed. To activate the enzyme, PQQ was added until saturation. The soluble fraction was incubated (53 °C, 1 h) and precipitated protein was removed by centrifugation. As a final step, a S-sepharose cation exchange chromatography column was equilibrated (20 mM Tris, 10 mM CaCl pH 7,5) and loaded with the supernatant from the previous step. The PQQ-dependent glucose dehydrogenase was eluted with a gradient from 0.1 to 1 mM NaCl. REIA as Sortase Activity Assay with GDH. Indicated concentrations of sortase or transglutaminase were mixed with assay buffer, 20 μM of glucose dehydrogenase containing one of the substrates of the reaction (LPKTG, glutamine or lysine and biotin (containing the other substrate of the reaction GGGG, lysine or NHS-ester)). This reaction mixture was incubated at 37 °C for 2 h (1 h for mTG and NHS-ester). The reaction was stopped by the addition of a 20-fold volume excess of inhibition buffer (50 mM Tris/HCl, 200 mM NaCl, 10 mM CaCl2, 5 mM iodoacetamide or 100 mM lysine, pH 7.5). The stopped reaction mixture was then centrifuged (5000 g, 25 °C, 10 min). The supernatant (50 μL) was added to 100 μL beads (SA-MAG) (20 mg/mL beads, 50 mM Tris/HCl, 200 mM NaCl, 10 mM CaCl2, 5 mg/mL BSA, 0.1% Triton X-100, pH 7.5) (The Streptavidin Magnetic Particals were purchased from Sigma-Aldrich (11641786001)). The mixture was incubated (30 °C, 200 rpm, 30 min). Thereafter the magnetic beads were washed five times with 300 μL washing buffer in V-bottom multiwell plates using a magnet and a vacuum pump. Afterward the beads were resuspended in 100 μL citrate buffer (200 mM, pH 5.8, 10 mM CaCl2) and 50 or 80 μL thereof was transferred to a fresh well. To that 150 μL test buffer (200 mM sodium citrate, 0.3 g/L (4-(dimethylphosphinyl-methyl)-2-methylpyrazolo-[1.5a]-imidazol-3-yl)-(4-nitrosophenyl)amine, 1 mM CaCl2, 30 mM glucose, pH 5.8) were added. Initial reaction rates of the reporter enzyme were measured over a time period of 5 min at 620 nm. REIA as High-Throughput Screening Assay in Crude Cellular Extract. Cells (E. coli BL21) expressing sortase were cultured in 96 well plates, containing 200 μL LB-media per cavity, overnight at 37 °C and 200 rpm. Each cell suspension was mixed 1:10 with lysis buffer (50 mM Tris/HCl, 0,1% TritonX-100, 200 mM NaCl, 5% B-PER, pH 7.5) and incubated (50 °C, 30 min). Lysate (50 μL) was then mixed with assay buffer containing 20 or 4 μM of glucose dehydrogenase-LPXTG and 20 μM GGG-biotin substrates and incubated (2 h, 37 °C). The reaction was stopped by addition of a 20-fold excess of inhibition buffer. The stopped reaction mixture was centrifuged (5000 g, 25 °C, 10 min). The supernatant (50 μL) was transferred to a streptavidin coated multiwell plate (SA-MTP) (from Microcoad 11643673) and 100 μL assay buffer was added and incubated 30 min (30 °C, 200 rpm). Thereafter the microtiter plate was washed eight times with 300 μL washing buffer. After washing, 150 μL test

Figure 6. (A) Sa-SrtA reaction of a LCRed640 labeled LPETG peptide with glycine labeled biotin, establishing a peptide bond between the biotin an the fluorophore. (B) Different Sa-SrtA concentrations were applied to adopted REIA conditions (20 μM tetra glycine-giotin, 20 μM LCRed640-LPETGGRR, in 50 mM Tris-HCL buffer pH 7.5, 200 mM NaCl, 10 mM CaCl). After 2 h the reaction was stopped, diluted, and transferred to a SA-MTP. The plate was washed and analyzed for fluorescent intensity Ex 620/10. Em 645/10.

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MATERIALS AND METHODS Solid-Phase Peptide Synthesis of Sortase Substrates. H-Gly4-Ebes-Lys(Bi)-OH and H-Ala4-Ebes-Lys(Bi)-OH were synthesized via standard Fmoc-based solid phase peptide synthesis in a 0.25 mmol scale using Fmoc-Lys(biotinyl)Wang resin (Bachem, D-2705), Fmoc-Ebes (Iris Biotech, PEG4970), and Fmoc-Gly-OH or Fmoc-Ala-OH (Iris Biotech, FAA1050). After solid-phase synthesis, the peptide was cleaved with TFA/water 20:1 and purified via RP18-HPLC using a water/TFA acetonitrile gradient. Peptides shown in Figure 1 and (SI, Table S1) were synthesized using standard Fmoc synthesis protocols and purified before labeling reactions were carried out. N-terminal labeling was achieved using aminoreactive dyes LCRed640 NHS ester or Cy5 NHS ester, respectively. Peptides containing lysine residues were labeled using BHQ NHS ester. Cysteine containing peptides were labeled using thiol reactive dye 5-iodoacetamidofluorescein. Sortase Expression and Purification. Cloning, overexpression, and purification of the wild-type Sa-SrtA Δ59 and P94S variant was performed according to the method of Kruger et al.14 In this study a pQE80L vector and BL21 E. coli cells were used for overnight expression in LB-media. For purification, the cells were lysed via French press and centrifuged. The supernatant was applied to a Ni-NTA resin and afterward to a Superdex 75 column. The final product was dialyzed against buffer (50 mM Tris/HCl, 200 mM NaCl, 10 mM CaCl2, pH 7.5). FRET Based Assay. Indicated concentrations of sortase and DMSO-reconstituted peptides were dissolved in assay buffer (50 mM Tris/HCl, 200 mM NaCl, 10 mM CaCl2, pH 7.5). A carry eclipse was used to analyze the samples, windows of 5 nm around the indicated wavelengths were used for excitation and emission. G


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buffer was added. Initial reaction rates of the reporter enzyme (glucose dehydrogenase) were measured over a time period of 5 min at 620 nm. REIA as Sortase Activity Assay with LCRed640. Indicated concentrations of sortase were mixed with assay buffer (50 mM Tris/HCl, 200 mM NaCl, 10 mM CaCl2, pH 7.5) and 20 μM of LCRed640 containing one of the substrates of the sortase reaction (LPETG) and biotin (containing the other substrate of the sortase reaction, GGGG)). This reaction mixture was incubated at 37 °C for 2 h. The reaction was stopped by the addition of a 20-fold volume excess of inhibition buffer (50 mM Tris/HCl, 200 mM NaCl, 10 mM CaCl2, 5 mM iodoacetamide, pH 7.5). The supernatant (50 μL) was added on to a SA-MTP (Nunc 384 white). The plate was incubated (30 °C, 200 rpm, 30 min). Thereafter, the microtiter plate was washed eight times with 100 μL washing buffer. After washing, fluorescence intensity was analyzed (Ex 620/10. Em 645/10).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00111. Overview of HTS-RAQ determination and supplementary figures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS All authors received funding from Roche Diagnostics GmbH. ABBREVIATIONS Abz, 2-aminobenzoyl; BHQ2, Black Hole Quencher-2; ChIP, Chromatin-Immuneprecipitation; Cy5, Cyanin 5; Dap, 2,4dinitrophenyl; FISH, fluorescent DNA In situ hybridization; FITC, Fluorescein isothiocyanate; FRET, Förster resonance energy transfer; HPLC, high performance liquid chromatography; MAG, magnetic beads; MS, mass spectrometry; mTG, microbial Transglutaminase; MTP, microtiter plate; NHS, Nhydroxysuccinimide; PQQ, pyrroloquinoline quinone; RAQ, relative affinity quotient; REIA, Reporter Immobilization Assay; Sa-SrtA, sortase A from Staphylococcus aureus; Sort-tag, recognition motive LPXTG (X = K or E in this study)

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REFERENCES

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