Team:TU Darmstadt/Project/Sortase

TU Darmstadt

Sortase

Introduction

Sortases are transpeptidases, most commonly found in gram-positive bacteria[1]. Since it was our ambition to create a system enabling customizable modifications on the surface of P22 Virus-like particles (VLP), [2] we chose sortases of class A, specifically Sortase A5M (BBa_K3187006) and Sortase A7M (BBa_K3187028). In their natural context, sortases of class A attach glycoproteins onto the surface of the cell. The reaction is initiated by covalently binding the C-terminal LPETG-motif of one substrate to the active site of the sortase. Sortase then cleaves in between the glycine and threonine of the LPETG sequence, before establishing a peptide bond connecting it to the N-terminal poly-glycine (GGGG) sequence. [1]

Sortase A5M is a catalytically more efficient variant of Sortase A from the gram-positive bacterium Staphylococcus aureus. It was generated via directed evolution and needs Ca2+ to work [3] . However, for our project we needed a variant that provides catalytic activity in living bacteria. Therefore, Sortase A7M was selected, being the first catalytically improved variant, that is not dependent on Ca2+. The calcium-independence of this variant enables applications for in vivo approaches of VLP production and surface modification [3] .

Since there was a Sortase A variant available in the registry, provided by the iGEM team Stockholm from 2016, we decided to characterize this part, as we were already using two different variants in our project.

Animation 1: 3D-Structure of Sortase A7M. To find out more, visit our modeling page.

Achievements

    Checkbox Enzyme kinetics of Sortase A7M/ Sortase A5M

    Checkbox Prooved Ca2+-independency of Sortase A7M for in vivo approaches

    Checkbox Bronze Criterion: Characterization Sortase A BBa_K2144008

    Checkbox Discovery of new superior FRET-pair

    Checkbox Providing the iGEM community with a well characterized enzyme Sortase A7M for protein engineering

Cloning

The methods used for cloning of the different mutants of the sortase were restriction and ligation via NdeI and SalI and Gibson assembly. The Sortase A5M was cloned into pET24(+)-vector via restriction and ligation with NdeI and SalI as restriction enzymes. The vector posesses a kanamycin resistance and is controlled through a T7 promoter, which can be induced with IPTG. Sortase A7M is controlled by the same T7 promoter. Sortase A, introduced by iGEM Stockholm 2016, was cloned via Gibson assembly into PSB1C3. This backbone has a chloramphenicol resistance and is also controlled under a T7 promoter. Cloning of all products was checked via sanger sequencing.

Expression and Purification

After successfully transforming our sortase genes in BL21 cells, we inoculated 100 mL overnight cultures, with the respective antibiotic. The next day 1 L cultures were inoculated with the overnight culture to reach OD600 = 0.1. Subsequently the cultures were incubated under constant shaking at 37 °C until they reached OD600 = 0.6. At OD600 = 0.6 the cultures were induced with 0.5 mL of 1 M Isopropyl-β-D-thiogalactopyranosid (IPTG). The gene expression was performed at 30 °C under constant shaking overnight. After expression of Sortase A7M, Sortase A5M, and Sortase A from Stockholm (BBa_K2144008) in BL21 cultures the cells were crushed via EmulsiFlex (Avestin) and proteins were purified through affinity chromatography via Fast Protein Liquid Chromatography (FPLC) with the ÄKTA pure (GE Healthcare, Illinois, USA). His-Tag was used for purification of Sortase A7M and Sortase A (Stockholm) and Strep-Tag II was used for purification of Sortase A5M.

Figure 1: Protein purification procedure at EmulsiFlex (Avestin) and ÄKTA pure (GE Healthcare, Illinois, USA)

SDS-PAGE

To verify the successful production of of Sortase A7M, Sortase A5M, and Sortase A BBa_K2144008, SDS-PAGEs were performed. The resulting bands were compared to the molecular weight of the different sortase variants. Also, SDS-PAGEs were completed to verify enzymatic activity in assays prior to measuring sortase properties via Fluorescence Resonance Energy Transfer (FRET).

Fluorescence Resonance Energy Transfer (FRET)

To determine the kinetics of our transpeptidase variants, FRET assays were performed in 384 well-plates (dark) using a Tecan plate reader. A FRET relies on the phenomenon that an excited fluorophore (donor) transfers energy to another fluorophore (acceptor), thereby exciting it. This process only works if both fluorescent molecules are in close proximity and depends on the FRET-Pair. By transferring the energy from donor to acceptor, the donor's emission is reduced and the intensity of the acceptors emission is increased [4] . The efficiency depends on the distance between the fluorophore, the orientation and the spectral characteristics [5] . You can see the principle of FRET in Fig. 2.

Figure 2: Jablonski diagram showing the energy transfer between a FRET-pair (inspired by Alex M. Mooney).

Mass Spectrometry (MS)

To estimate the product yield of catalyzed reactions by Sortase A7M we performed mass spectrometry. The tested molecules can be distinguished between products and educts due to desorption and ionization. Therefore, we used the electrospray ionization (ESI) technique for the mass spectrometry. This technique has a low resolution but is a very soft ionization method, which makes it an optimal method for biological molecules. [6]

Enzyme-linked Immunosorbent Assay (ELISA)

The enzyme-linked immunosorbent assay (ELISA) is an analytical assay frequently utilized for immobilization and verification of different macro molecules. Immobilizing the recognition tag of the sortase on a surface allows us to verify the coupling efficiency of Sortase A7M under certain conditions. Firstly, we functionalized paper to presenting a poly-G peptide sequence on the surface. Using the Sortase A7M, a ZZ-domain carrying a LPETG amino acid sequence is coupled to the short peptide sequence of GGGßA. The ZZ-domain itself shows high affinity to the human IgG antibody FC-domain and therefore allows the following immobilization of IgG [7] . The secondary antibody is an anti-human IgG, Fab-specific antibody carrying the horseradish peroxidase enzyme (HRP). HRP is capable of converting 4-chloro-1-naphthol to benzo-4-chlorocyclohexandienone using hydrogen peroxide. This color-reaction allows us to draw a conclusion about the previous ZZ-domain’s sortase-mediated coupling efficiency since the turnover of the HRP is directly connected to the ligated ZZ-domains.

To get some more detailed descriptions of these methods, take a look at our notebook.

Characterization of Sortase A7M and Sortase A5M

How do we validate if our purified sortases are active?

After purification of the sortases, we first performed SDS-PAGEs to verify that they are pure and monomeric. You can see in Fig. 3 that the purifications were successful. Next, we tested if the purified sortases connect two proteins that carry the important Sortase-recognition tags, N-terminal polyG and C-terminal LPETGG. Therefore, we added the sortases to a mix of GGGG-mCherry and mCherry-LPETGG. The reactions were performed in different buffers, at different enzyme-to-substrate ratios and for different time spans. We performed an SDS-PAGE, and prior to Coomassie staining, we recorded fluorescent images of the gel. Thereby, we could identify mCherry bands in the gel.

Figure 3: SDS-PAGE of Sortase A7M and Sortase A5M where the bands show up at approximately 15 kDa. Our estimated size for Sortase A7M was 17.85 kDa, and for Sortase A5M 18.07 kDa. This confirms the result shown on the gel, since the band of Sortase A5M is a little higher than the one of Sortase A7M.

Figure 4:

a) Fluorescence gel of the sortase-reaction of GGGG-mCherry and mCherry-LPETGG mediated by Sortase A7M incubated for 2 h and 4 h each. Reaction solutions were mixed with different ratios from enzyme to substrate concentration(1:3;1:10) and each incubated in two different buffers (Tris-HCl and Ammoniumdicarbonat). Product bands at a height of about 57 kDa can be seen in lane 4, 5, 6, 8, 9 (from left to right). The bands below the product at about 38 kDa could be semi-denatured mCherry dimers. b) Fluorescence gel on top of the Coomassie-stained gel of the sortase-reaction of GGGG-mCherry and mCherry-LPETGG mediated by Sortase A7M incubated for 2 h and 4 h each. Reaction solutions were mixed with different ratios from enzyme to substrate concentration(1:3;1:10) and each incubated in two different buffers (Tris-HCl and Ammoniumdicarbonat). Product bands at a height of about 57 kDa can be seen in lane 4, 5, 6, 8, 9 (from left to right). The bands below the product at about 38 kDa could be semi-denatured mCherry dimers. Additionally, Sortase A7M can be seen at 17 kDa.The unprocessed mCherry monomers can be seen at 28 kDa.

As shown in Fig. 4, under certain conditions, a product band appeared at the expected size of 57.3 kDa (28.5+28.8 kDa). From this first activity test, we draw three conclusions:

  • Our purified Sortase A7M is active
  • The enzyme-substrate ratio affects the product yield
  • The duration of the reaction affects the product yield


Additionally, TRIS buffer seems to alter the Coomassie staining efficiency of Sortase A7M. This endpoint measurement gave us a first impression that our Sortase A7M works nicely. Of course, we wanted to further characterize the parameters of the reaction. When we understand the Sortase better, modification of our VLPs will become more straightforward.

How do we measure sortase reaction kinetics?

In the above described assays, we noticed the impact of enzyme-substrate ratio and reaction duration on the overall product yield. We thought about how to further measure the kinetics of the sortase reaction. In the literature, sortase reaction kinetics are often measured by FRET-assays. Therefore, we designed a suitable FRET-assay. In the end, we came up with a new FRET-pair not described in the literature to date: of 5-Carboxytetramethylrhodamin-LPETG (TAMRA-LPETG) and GGGG-sfGFP. If you like to know more about our journey towards this superior FRET-pair, just open the following box.

Discovery of a new FRET pair

For characterization of the reaction kinetics of Sortase A7M, Sortase A5M and Sortase A BBa_K2144008, we decided to develop a suitable FRET pair.

In order to find an optimal FRET pair, we first recorded an emission and absorption spectrum of TAMRA-LPETG and GGGG-mCherry to verify the suitability for the FRET effect, checking for a possible overlap between the donor's emission and the acceptor's excitation.

Figure 5: Establishment of a FRET-pair of 5-TAMRA-LPETG (TAMRA) and GGGG-mCherry (mCherry). In this configuration TAMRA acts as donor and mCherry as acceptor. When the two fluorophores are not linked via the substrates of the sortase only TAMRA is being excited. After sortase-mediated ligation of the two substrates mCherry is the fluorophore being excited via the FRET and the emission of mCherry intensifies. Meanwhile, the emission of TAMRA decreases

TAMRA is a chemical fluorophore that has an absorbance maximum at 543 nm and an emission maximum at 570 nm[8]. The terminal carboxy group of the dye was linked via a lysine linker to the LPETG sequence (see Fig. 5). mCherry has an N-terminal poly-glycine sequence and can therefore be linked to the LPETG motif of TAMRA via the Sortase A. For a sufficient FRET-effect, it is also necessary that the distance between donor and acceptor is lower than the Förster radius. The Förster radius describes the distance between two fluorophores at which 50 % of the energy is transferred [5] .

First, we wanted to identify which concentrations are needed for our experiment, then set up the reaction and measured fluorescence intensities. Over time, a decline in the emission of TAMRA can be observed as Sortase A7M/A5M is converting more educts to products.

Figure 6: The graph shows the excitation and emission spectra of TAMRA and mCherry. Due to the large overlap of TAMRA emission and mCherry excitation it is possible to perform a FRET with this pair of fluorophores. The graph show the relative fluorescence unit (RFU[%]) in relation to the excited/emitted wavelength [nm]. The peaks are normalized to 100 %.

The emission and excitation spectra of TAMRA and mCherry exhibit an overlap of emission of TAMRA and excitation of mCherry. Based on this output, a FRET-assay for the kinetics of Sortase A7M was performed to confirm whether the FRET-pair is working. As TAMRA is excited with light of a lower wavelength than mCherry, the former serves as FRET donor and the latter as acceptor. We chose the excitation wavelength at 485 nm to prevent unnecessary “leak” excitation of mCherry. Nevertheless, an excitation of mCherry could not be excluded and may have negative effects on the visibility of the FRET.

Figure 7: Spectrum of the negative control of TAMRA and mCherry, without Sortase A7M, over the course of 20 min in 5 min intervals. Depicted are the emission wavelengths against the RFU.

Figure 8: Spectrum of TAMRA and mCherry, with Sortase A7M, over the course of 20 min in 5 min intervals. Depicted are the emission wavelengths against the RFU. The sortase-mediated ligation results in a decline of both emission peaks.

The analysis of the data shown in Fig. 7 confirmed the aforementioned suspicion that mCherry is also excited at 485 nm, which makes differentiation of the fluorescence more difficult. Furthermore, Fig. 8 shows that the difference in the decline of TAMRA is not significant. Accordingly, a decline in the emission maximum of TAMRA over time is also visible in the negative control. One reason might be bleaching of TAMRA through the excitation by the laser. Nevertheless, conversion by the Sortase A7M can be observed by comparing the results with the negative control.

Figure 9: Sortase reaction in TAMRA-mCherry-FRET after subtracting the negative control. Depicted is the difference in RFU over time [min]. Within the first 20 min of the substrate conversion is the quickest. At 30 min a plateau is reached. After 60 min starts catalyzing the reverse reaction. The mean ΔRFU value was normalized to zero for better visualization. The ΔRFU refers to the negative control without the respective sortase at 570 nm.

To confirm the functionality of the Sortase A7M, another more sufficient FRET-pair was developed. The measured absorbance and emission spectra indicated that TAMRA and superfolder green fluorescence protein (sfGFP) are a possible FRET-pair. The sfGFP has an N-terminal polyglycine sequence and can therefore be linked to TAMRA with the sorting motif, in the same way as mCherry was connected. However, the small overlap between the excitation spectra of sfGFP and TAMRA could solve the previous “simultaneous excitation” problem we observed for the mCherry-TAMRA FRET-pair. Because of the lower excitation maximum of sfGFP compared to TAMRA, sfGFP was chosen as donor and TAMRA as acceptor. sfGFP was excited at 465 nm to minimize the unnecessary leak excitation of TAMRA.

Figure 10: The graph shows the excitation and emission spectra of TAMRA and sfGFP. Due to the large overlap of sfGFP emission and TAMRA excitation it is possible to perform a FRET with this pair of fluorophores. The graph show the relative fluorescence unit (RFU[%]) in relation to the excited/emitted wavelength [nm]. The peaks are normalized to 100 %.

Figure 11: Design of a FRET-pair of 5-TAMRA-LPETG (TAMRA) and GGGG-sfGFP (sfGFP). In this configuration sfGFP acts as donor and TAMRA as acceptor. When the two fluorophores are not linked only sfGFP is being excited. After sortase-mediated ligation of the two substrates, TAMRA is the fluorophore being excited via FRET and the emission of TAMRA intensifies. Meanwhile, the emission of sfGFP decreases.

The transfer of energy from sfGFP to TAMRA can be seen by the decrease in emission of sfGFP and an increase in emission from TAMRA. Compared to TAMRA as an acceptor, the sfGFP bleaches significantly less and is consequently more suitable as a donor for FRET. Furthermore, the afore mentioned problem of simultaneous donor and acceptor excitation seems to be solved. It seems that we have found a FRET-pair with superior properties.

Figure 12: Spectrum of the negative control of TAMRA and sfGFP, without Sortase A7M, over the course of 25 min in 5 min intervals. Depicted are the emission wavelengths against the RFU.

Figure 13: Spectrum of TAMRA and sfGFP, with Sortase A7M, over the course of 25 min in 5 min intervals. Depicted are the emission wavelengths against the RFU. The sortase-mediated ligation results in a decline of both emission peaks.

Due to the collected data of both FRET-pairs we decided to use the TAMRA-LPETG and GGGG-sfGFP FRET-pair for further characterization of our Sortase A variants. Two reasons justify this decision:

  • TAMRA bleaches stronger than sfGFP when excited with a laser.
  • The spectral overlap between TAMRA and mCherry disturbs “clean” energy transfer, thus, making the FRET-effect less visible and could not be used for analysis of the sortase-mediated reaction.

For the recording of sortase reaction parameters we recommend using the FRET-pair sfGFP-TAMRA. As this pair of fluorophores proved to have near perfectly aligned spectra and since the bleaching effect is visibly lower on sfGFP than on TAMRA, we chose to use this FRET-pair in most of our following assays. Nevertheless, we do not rule out the use of TAMRA-mCherry as a FRET-pair since we used it in several FRET-assays as well.

Animation 2: Animation of Sortase A7M enzyme kinetics over the course of 3 h. The reaction speed increases radically in the beginning moving from RFU 8000 to RFU 6000 at λ = 550 nm where a plateau is reached (green). The negative control (red) is also reduced in its RFU due to bleaching. Nevertheless, a peak at λ = 580 nm arises already after short reaction time. This peak indicates the successful Fluorescence Resonance Energy Transfer.

Why are enzyme-substrate ratio and duration important for the sortase reaction?

In one of our first FRET experiments, we addressed the simple theory: More sortase in the reaction mix improves the initial product formation. For this, we used the TAMRA-LPETG : GGGG-mCherry FRET pair. We measured the FRET change over time in a multiwell platereader (Fig. 14).

Figure 14: Reaction kinetics of Sortase A7M in different concentrations at same level of substrate concentration (6 µM TAMRA-LPETG and (M)GGGG-mCherry). The light blue graph shows the reaction when the sortase concentration is higher (5 µM) and lower concentration (1 µM), in the dark blue graph. Wen the enzyme concentration is lower the maximum in substrate conversion is reached later. The light blue graph also shows a slight decline in product concentration from 60 min onwards.The ΔRFU refers to the respective negative control without each sortase at 570 nm. The mean ΔRFU value was normalized to zero for better visualization.

These observations underline that the higher the sortase concentration, the faster the reaction and product formation.

However, in this assay we observed a striking feature of the sortase reaction. In the reaction with more Sortase A7M present, the FRET change started to decrease after a certain maximum was reached! We suspected some kind of dead-end product formation, as the sortase does also catalyze the reverse reaction of product to educts. Therefore, the overall reaction duration is a very important parameter. We gathered more details about the role of the reverse reaction during our comparison of Sortase A7M and Sortase A5M. Just keep reading if you want to know more!

Who wins – Sortase A7M or Sortase A5M?

In our introduction we described that Sortase A7M and Sortase A5M are both fascinating enzymes, although each of them has a unique „selling point“. Sortase A5M is faster, whereas Sortase A7M is Ca2+-independent. We confirmed both of these points in extensive FRET-assays. According to the literature, Sortase A5M works best with a Ca2+-concentration of 2 mM. In contrast, Sortase A7M is a calcium-independent mutant of the enzyme. Moreover, Ca2+ even seems to inhibit this enzyme variant slightly [1].

Firstly, we confirmed that in contrast to Sortase A5M, Sortase A7M is Ca2+-independent. The results are shown in Fig. 15 Sortase A7M also works in presence of Ca2+, but these FRET experiments made us suspect that Ca2+ may even inhibit Sortase A7M.

Figure 15: Sortase A7M FRET-assay of connecting TAMRA-LPETG with GGGG-sfGFP with and without Ca2+. The Sortase A7M reaction was measured with 6 mM Ca2+ every minute. Sortase A7M reaction without Ca2+ was measured every three minutes. It is shown that this enzyme variant works with calcium and without calcium as well, although it seems like Sortase A7M is slightly inhibited due to the presence of calcium which explains why green graph is lower than the red one. The ΔRFU refers to the respective negative control without each sortase at 514 nm. The mean ΔRFU value of the duplicates was normalized to zero for better visualization.

Secondly, we confirmed that Sortase A5M is inactive if Ca2+ is absent, which can be seen in the right graph in Fig. 16. As expected, Sortase A5M shows increasing enzymatic activity with increasing Ca2+ levels. The reaction runs fastest with 2 mM Ca2+, and the maximal FRET change (in terms of ΔRFU) is reached after 37.5 min. Strikingly, the FRET change decreases afterwards. We observed this phenomenon before and assume this to be due to dead-end product formation caused by the reverse reaction.

Figure 16: Both figures show Sortase A5M FRET assay connecting TAMRA-LPETG and GGGG-sfGFP with different Ca2+-concentrations. The right graph is showing that Sortase A5M does not work without calcium. The negative ΔRFU measurements are probably caused by measuring errors by the Tecan platereader. The ΔRFU refers to the respective negative control without each sortase at 514 nm. The mean ΔRFU value of the duplicates was normalized to zero for better visualization.

According to the results of this assay, Sortase A7M is definitely Ca2+-independent, since it shows linking activity without calcium in the vicinity. The enzyme mutant also works in presence of Ca2+ (Fig. 15), but these FRET experiments made us suspect that Ca2+ may even inhibit Sortase A7M, since it shows less activity with calcium around than without calcium.

To better address this question, an ELISA was performed. Therefore, a piece of paper functionalized with GGG-Beta-Alanin (GGGβA) was connected to a protein domain, which binds antibodies to the LPTEG-tag. The results are shown in Fig. 17.

Figure 17: Absorbance at 450 nm at a temperature of 23.8˚ C
In well 1 additional 10 mM Ca2+ were added which was not the case in well 2. Well 3 serves as a negative control since the enzyme is missing in this reaction.

As shown in Fig. 17, the highest absorption was measured in well 2. Thus, Sortase A7M works more efficiently when no Ca2+ is around. The absorption is also relatively high for the negative control, which can be explained by poor washing before the substrate for Horseradish peroxidase (HPR) was added. This assay shows the functionality of Sortase A7M even in context of surfaces since we confirmed that Sortase A7M is able to connect tags attached to paper. This shows that the surface structure is not a relevant factor for the enzyme.


Figure 18: Comparison of the reaction speed of Sortase A5M with Ca2+ and Sortase A7M without Ca2+, therefore each working under optimal conditions. The kinetics were measured via a FRET connecting TAMRA-LPETG and GGGG-sfGFP. The ΔRFU refers to the respective negative control without each sortase at 514 nm. The mean ΔRFU value of the triplicates was normalized to zero for better visualization.

When we compare the reaction speed of Sortase A5M and Sortase A7M, Sortase A5M is the clear winner (see Fig. 18). However, this means of course that the reverse reaction is also faster in the case of Sortase A5M. Consequently, Sortase A7M is the best variant for in vivo modification of our VLPs as it is Ca2+-independent. On the other hand, Sortase A5M is a suitable enzyme variant for in vitro modification due to its high efficiency.

What about other substrates?

Primary Amines

When we consider the future of our MVP, we or other iGEM teams may want to attach other molecules than proteins to VLPs, maybe even directly in E. coli. The literature describes Sortase A7M as somewhat „promiscuous“ towards other substrates than GGGG(polyG) as long as the substrate possesses a primary amine [9] . To confirm this, we performed additional assays with other substrates in the lab of Prof. Kolmar. The Sortase A7M used for this assay was stored in the fridge at 4 °C for two weeks. The substrates were TAMRA with a KLPETG bound to TAMRA via the lysine side chain and 3-azidopropanamine as the example for a primary amine. The reaction was performed for two hours at 37 °C. It was then analyzed by electron spray ionization mass spectrometry (ESI-MS) (Fig. 19).

Figure 19: Mass spectrum before the reaction of TAMRA-LPETG with 3-azidopropanamine showing the educt at 1054 g/mol.

Fig. 19 shows the educt-peak in the mass spectrum. TAMRA with the LPETG-tag weighed 1054 g/mol. Shown above in green is the single charged molecule at 1054.27 g/mol and the double charged molecule at 528.75 g/mol.

Figure 20: Mass spectrum after the reaction of TAMRA-LPETG with 3-azidopropanamine showing the product at 1079.37g/mol.

Fig. 20 shows the product-peak in the mass spectrum. The primary amine that was taken as an example has a molecular weight of 100 g/mol. After the reaction the glycine of the LPETG-tag has been removed and therefore the product only consists of TAMRA-KLPET-3-azidopropanamine. When adding the two molecular weights and subtracting the weight of the glycine it adds up to a total weight of 1078 g/mol which can be seen in the single charged 1079.37 g/mol peak (Fig. 19), since the ESI-MS we used has a small error margin. The peak in black again is the double charged peak at 541.55 g/mol. This clearly shows that the sortase reaction took place. Furthermore, we can conclude that the Sortase A7M accepts any primary amines as a substrate. However, the mass spectrum does not show the ratio of educt and product, which is why we cannot estimate whether the turnover is as high as when using a polyG-tag as substrate. Additionally, this assay confirms our suspicion that the Sortase A7M is stable at 4 °C and still functional if stored at said temperature for at least two weeks.

Yield

For the characterization of Sortase A7M an assay was designed to show the coupling efficiency between the TAMRA-LEPTG and the tetrapeptide GGGβA catalyzed by the Sortase. The β-Alanin functions as a spacer. The Sortase reaction was performed for 1h at 30 ˚C and was stopped by enzyme separation through centrifugal filtration. For analysis ESI-MS was used. The mass spectrometry enables differentiation between products and educts. It allowed us to make an estimate of the product yield. The calculated theoretical molecular masses are 1054 g/mol for TAMRA and 1240 g/mol for TAMRA-LPETGGGβA. Therefore, peaks are expected at mass/n, with n ∈ N. By comparison of the number of corresponding peaks, estimation of the product yield is possible as both molecules possess the same amount of ionizable groups and thus the difference in the ionizability of both molecules is negligible.


Figure 21: Mass spectrum of the sortase-mediated ligation of TAMRA-LPETG and GGGβA showing the difference in height of the educt-peak and the product-peak which can be used to estimate the yield of our Sortase A7M.


In Fig. 21 the 621.56 peak can be assigned to the TAMRA-LEPTGGGβA and the 528.85 to the TAMRA-LPETG. The count ratios of the two molecules mentioned show an excess of the product.

Is Sortase A7M able to attach cargo to P22 Coat protein?

One of the most important questions in our project was: Can Sortase A7M attach proteins to tagged P22 coat protein (CP-LPETGG)? This is one of the absolute requirements of our MVP platform.

We performed the linking reaction with CP-LPETGG and GGGG-mCherry as substrates and applied them to an SDS-PAGE. We saw products at the expected size (28 kDa + 49 kDa = 77 kDa) thus the requirement is fulfilled. However, a lot of additional bands appeared that we did not expect. These bands also appeared when only Sortase A7M and CP were mixed.

Figure 22: a) Sortase A7M band is at expected height (17.85 kDa). The two negative controls containing only GGGG-mCherry (28 kDa) and CP-LPETGG (49 kDa) at the expected respective heights. b) Shown are sfGFP-SP and CP-LPETGG each incubated with both Sortase A7M and Sortase A5M. Both gels display multimers when coat protein and a sortase variant are in a sample together.

To investigate this issue, we had a look at the literature and found a matching description in the publication of Patterson et al. They performed a similar experiment with P22 capsid proteins and observed the same multimers in their SDS-PAGEs [2] . Comparing both SDS-PAGEs, we came to the following assumption:

Because of the promiscuity of Sortase A7M to accept primary amines as substrates, as we discussed previously, the formation of CP multimers occurs, unspecifically catalyzed by Sortase A7M.

Parallel to these experiments, we successfully modified the exterior of pre-assembled VLPs in vitro (VLP assembly). These modified VLPs were homogenous and overall correctly assembled. Therefore, we conclude that the described multimer problem only occurs when Sortase A7M encounters free CP.

Does methionine affect Sortase linking?

Sortase A7M preferably attaches N-terminal poly-G to C-terminal LPETGG. However, the first amino acid of a protein is methionine (to be specific, formylmethionine in bacteria). For our constructs that possess N-terminal polyG-tags, we have to ask ourselves the question: If the initial methionines are not cleaved off after the proteins have been produced, will this interfere with the Sortase reaction?

To investigate this, we cloned and purified two other proteins: TVMVsite-GGGG-mCherry and TEVsite-GGGG-sfGFP. Then we treated these proteins with the respective proteases, resulting in *GGGG-mCherry and *GGGG-sfGFP. Following this *GGGG-mCherry was then compared to (M)GGGG-mCherry which we used in all previous assays. Assays were also conducted on Fig. 23 the processed *GGGG-sfGFP substrate. Fig. 23 confirmed our assumptions that the unprocessed substrate cannot be linked to the sorting motif via Sortase A7M. Subsequently, *GGGG-sfGFP (after protease digest) demonstrate successful linkage via sortase-mediated ligation.

Due to these findings we modified our VLPs with *GGGG-sfGFP.

Figure 23: Sortase-mediated ligation of TAMRA-LPETG and GGGG-sfGFP (with TEV cleavage site) one cut with TEV protease and one not. The sample with the unprocessed substrate shows no increase in RFU. In contrast the processed substrate shows a clear increase in ΔRFU. After 90 min the reverse reaction begins.The ΔRFU refers to the respective negative control without each sortase at 514 nm. The mean ΔRFU value of the duplicates was normalized to zero for better visualization.

We performed FRET-assays with TAMRA-LPETG and either of the following reaction partners:

  • (M)GGGG-mCherry, a protein sample that might still carry an N-terminal methionine
  • *GGGG-mCherry that does not carry any additional N-terminal residue

Before the FRET-assay was started, we adjusted the mCherry-concentrations of both fluorescent protein solutions to the same level. To do so, we diluted them until both showed the same fluorescence at 610 nm.

Figure 24: FRET of the sortase reaction connecting TAMRA-LPETG and GGGG-sfGFP mediated by Sortase A7M. The concentration of the Sortase A7M and TAMRA-LPETG was kept at the same level why the concentration of sfGFP was either 10,7 µM or 1,4 µM. The graphs show that the reverse reaction happens earlier if the GGGG-substrate concentration is lower. The ΔRFU refers to the respective negative control without each sortase at 514 nm. The mean ΔRFU value of the duplicates was normalized to zero for better visualization.

Strikingly, only the (M)GGGG-mCherry construct showed a clear decrease in ΔRFU after the maximum ΔRFU was reached (at about 160 min).

Figure 25: Sortase-mediated ligation of TAMRA-LPETG and GGGG-mCherry one cut with TVMV protease and one with a methionine infront of the GGGG-tag. As visible the reverse reaction happens earlier if the methionine is not cleaved of the GGGG-tag. The ΔRFU is referring to the negative controls without Sortase A7M at 570 nm. The mean ΔRFU value of the tripicates was normalized to zero for better visualization.

We assume the following: Although we adjusted the overall mCherry concentration by fluorescence, we cannot determine the absolute amount of MGGGG-mCherry in the (M)GGGG-mCherry sample. However, if this amount was relatively high, the effective substrate concentration that could enter the sortase reaction would be low. That is because (M)GGGG is a worse sortase substrate than GGGG – if any at all. If we furthermore consider that a low substrate concentration correlates with a faster reverse reaction, we can explain the observed decrease in ΔRFU for the (M)GGGG-mCherry sample that contrasts the ΔRFU trend of the *GGGG-mCherry sample.

On this basis we can assume that a certain, yet unknown portion of the (M)GGGG-mCherry sample still carries an N-terminal methionine.

These FRET-assays let us assume that methionine disturbs or at least interferes with the sortase reaction mechanism. Indeed, our modeling suggests that methionine affects the interaction of polyG and the flexible loop near the active site of Sortase A7M. Click here if you want to know more about our modeling results!

We propose that potential users of our platform introduce a protease cleavage site in front of the GGGG-protein in order to ensure successful modification of the VLP surface.

This strengthens our hypothesis: If there is any amino acid in front of the poly-glycine sequence, substrate binding to Sortase A7M is negatively influenced.

Are there other Sortases that might be useful?

Besides the Sortase A7M we used for modifying the VLPs we wanted to test other variants of this enzyme, such as the Sortase A5M. As we also wanted to further characterize a part of another iGEM team we took the opportunity to compare our mutant, Sortase A7M, with another Sortase, the Sortase A BBa_K2144008 which was provided by iGEM team Stockholm 2016. The only information we had on this Sortase variant, was its origin in the Sortase A-family and its calcium dependency.

To compare the different sortases, we expressed Sortase A7M, Sortase A5M and Sortase A from the iGEM team Stockholm 2016 (BBa_K2144008). As done with the Sortase A7M and the Sortase A5M, we purified the Sortase A (BBa_K2144008) via fast protein liquid chromatography (FPLC) using the ÄKTA pure (Fig. 26) by means of His-tag purification.

Figure 26: Chromatogram of the ÄKTA run. The elution of other proteins than Sortase A is shown.

Figure 27: Chromatogram of the ÄKTA run. Sortase A (BBa_K2144008) eluted as a peak at about 59 mL.


In Fig. 26 it is shown that the Sortase A (BBa_K2144008) eluted at about 59 mL. Fig. 27 shows the elution of other proteins than the Sortase A.

We started characterizing Sortase A BBa_K2144008 by inspecting the size of enzyme via a simple SDS-PAGE (Fig. 28).

Figure 28: A SDS-PAGE of a triplicate of the reaction solution for the sortase reaction consisting of coat protein (CP), mCherry and Sortase A BBa_K2144008. the components were mixed and incubated for 90 minutes at 37 °C. A negative control without Sortase A BBa_K2144008 in a triplicate is also included. To verify the size the Sortase A BBa_K2144008 was also put as a control on the gel solely. The depicted gel was edited to increase the contrast in order to show the Sortase A bands properly.

In Fig. 28 a triplicate of the Sortase A BBa_K2144008 is shown that indicates that the Sortase A BBa_K2144008 has a molecular weight of around 15 kDa which is what we had estimated from the sequence we received. The double band on the SDS-PAGE resulted from the solvent front appearing under the band of the Sortase A BBa_K2144008.
This gel also includes the various proteins, Sortase A7M, GGGG-mCherry and Coat-LPETGG after the sortase reaction that was run for 90 minutes at 37 °C. Conspicuously, the first two triplicates look the same, independent of whether the Sortase A BBa_K2144008 was in the reaction, or not. The first triplicate should show positive bands, at a height of about 74 kDa having 10 mM calcium in the buffer, but instead it shows the same outcome as the negative control without Sortase A BBa_K2144008. This raised the possibility that this Sortase variant might not work. To confirm our suspicion, we tested our Sortase A7M and the Sortase A BBa_K2144008 in a FRET-assay using TAMRA-LPETG and GGGG-sfGFP as substrates (Fig. 29). We ran the measurement for 3 h at 30 °C.

Figure 29: A FRET-assay of Sortase A BBa_K2144008 and Sortase A7M connecting TAMRA-LPETG and GGGG-mCherry with and without 10mM calcium. The ΔRFU refers to the respective negative control without each sortase at 514 nm. The mean ΔRFU value of the triplicates was normalized to zero for better visualization.

As visible in Fig. 29 the Sortase A BBa_K2144008 does not show any activity during the reaction although 10 mM calcium was present in the reaction buffer. In contrary, the Sortase A7M, incubated without calcium, shows the expected increase in fluorescence visible in the ΔRFU at 514 nm over time. The Sortase A BBa_K2144008 does not seem to catalyze any reactions inspected by us. This confirms the SDS-PAGE that showed the same outcome, of the Sortase BBa_K2144008 being not functional.

Conclusion

Looking at all the results above we can conclude that the Sortase A7M altogether poses a very fitting enzyme for our desired use. This variant is calcium-independent and therefore well suited for in vivo applications. Accordingly, it perfectly fits our model of the genetic circuit since it is expressed in the cells to modify the VLPs even at low calcium concentrations. Finding the appearance of multimers in solutions of singled CP with sortase present, supports our thought of expressing Sortase A7M after the VLP assembly. As we managed to thoroughly measure the enzyme kinetics of all of our sortases we were able to compare our Sortase A7M with other mutants like the Sortase A5M. This comparison showed that the Sortase A5M is faster than the Sortase A7M if measured each at the optimal calcium concentration. Therefore, it poses a valuable option for in vitro modifications. All in all, Sortase A7M is a good choice for the modification of VLPs as one of our assays also shows that it is possible to attach non-proteogenic molecules to the LPETG-motif as long as it has a primary amine accessible. This poses a very high modularity of the MVP platform.

References

  1. ↑ Hee-Jin Jeong, Gita C. Abhiraman, Craig M. Story, Jessica R. Ingram, Stephanie K. Dougan; Generation of Ca2+-independent sortase A mutants with enhanced activity for protein and cell surface labeling; PlosOne 12; 4.12.2017 [1]
  2. ↑ Dustin Patterson, Benjamin Schwarz, John Avera, Brian Western, Matthew Hicks, Paul Krugler, Matthew Terra, Masaki Uchida, Kimberly McCoy, and Trevor Douglas, Sortase-Mediated Ligation as a Modular Approach for the Covalent Attachment of Proteins to the Exterior of the Bacteriophage P22 Virus-like Particle, Bioconjugate Chemistry, 2017 [2]
  3. ↑ Zhimeng Wu, Haofei Hong, Xinrui Zhao and Xun Wang; Efficient expression of sortase A from Staphylococcus aureus in Escherichia coli and its enzymatic characterizations; Bioresources and Bioprocessing; 18.02.2017; [3]
  4. ↑ Paul R. Selvin, The renaissance of fluorescence resonance energy transfer, Nature America, September 2000 [4]
  5. ↑ Forster, T. Discuss. Faraday Soc. 27, 7–17 (1959) [5]
  6. ↑ Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM; Electrospray ionization for mass spectrometry of large biomolecules; Science (New York, N.Y.), 1989 [6]
  7. ↑ Y. Nakamura S. Shibasaki M. Ueda A. Tanaka H. Fukuda A. Kondo, Development of novel whole-cell immunoadsorbents by yeast surface display of the IgG-binding domain, Applied Microbiology and Biotechnology, November 2001 [7]
  8. ↑ Sigma-Aldrich, Data sheet: 5-Carboxytetramethylrhodamine [8]
  9. ↑ Glasgow JE, Salit ML, Cochran JR (2016) In vivo site-specific protein tagging with diverse amines using an engineered sortase variant. J Am Chem Soc 138:7496–7499. [9]
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