Assemblase Scaffold
Laboratory Objective #1
Produce the Assemblase Scaffold
This is the first of our three laboratory objectives. We aim to produce and synthesise the assemblase scaffold. This product is utilised in Laboratory Objectives #2 and Laboratory Objectives #3
Figure 1. Schematic of Assemblase scaffold fused with enzymes. Assemblase is a heterohexameric complex composed of 2 units of αPFD-SpyCatcher subunits (orange) and 4 units of βPFD-SnoopCatcher (yellow). Enzymes fused with SpyTag or SnoopTag (grey) can spontaneously form covalent bonds with their respective Catcher, allowing for co-localisation.
Assemblase is a novel protein scaffold that specifically co-localises enzymes in a modular fashion. It is made up of three main components: a scaffold backbone, the enzymes, and an attachment system to link these two together.
The scaffold backbone is a heterohexamer composed of two alpha and four beta prefoldin subunits. Prefoldin is a highly thermostable protein chaperone isolated from archaea. These prefoldin subunits are fused to ‘catcher’ proteins called Spy and SnoopCatcher, yielding αPFD-SpyCatcher and βPFD-SnoopCatcher. These catchers covalently bind SpyTag and SnoopTag in a modular fashion.
We fused SnoopTags and SpyTags to our enzymes of interest, LXYL-p1-2 and DBATG38R/F301V, that participates in a two-step reaction to produce the chemotherapeutic drug, Paclitaxel.Objective
This section aims to achieve the following:
- To express and purify αPFD-SpyCatcher and βPFD-SnoopCatcher subunits.
- To show that αPFD-SpyCatcher and βPFD-SnoopCatcher subunits self-assemble into a hexameric structure.
- To demonstrate that Spy/SnoopTag containing proteins are able to conjugate to the Spy/SnoopCatchers on the prefoldin arms.
- To show the hexameric Assemblase scaffold binds to the enzymes of interest.
- To show that enzymes are functional and work more efficiently co-localised than being free in solution.
Summary of our findings
| Successes | Successfully expressed and purified αPFD-SpyCatcher and βPFD-SnoopCatcher subunits |
|---|---|
| Successfully conjugated mCerulean3-SnoopTag to the βPFD-SnoopCatcher | |
| Successfully conjugated mVenus-SpyTag to the αPFD-SpyCatcher | |
| Successfully demonstrated the assembly of αPFD-SpyCatcher and βPFD-SnoopCatcher into a hexameric structure | |
| Current Tasks | Demonstrate the utility of the αPFD-SpyCatcher and βPFD-SnoopCatcher using FRET |
| Conjugate pathway enzymes to the assemblase scaffold | |
| Future Directions | Perform enzymatic assays to determine the product turnover rate of enzymes attached to the Assemblase scaffold |
Results
Cloning
We obtained E. coli cells containing αPFD-SpyC and βPFD-SnoopC gene constructs assembled into pET19b plasmid from the 2018 UNSW iGEM team. More information on the design of the gene constructs can be found on the 2018 UNSW iGEM page.
Protein Expression and Purification
Protein expression assay
Cells containing αPFD-SpyCatcher or βPFD-SnoopCatcher plasmid genes were grown up in a large-scale overnight culture of Luria Broth containing ampicillin at 37°C. Once OD600 reached 0.6, protein expression was induced using 1 mM IPTG and grown for 20 hrs at 20°C. A protein expression assay was performed on a sample of the overnight culture, using a cell-lysis detergent (BugBuster) to separate soluble and insoluble proteins (Figure 2).
Figure 2. Protein expression assay using BugBuster to determine expression of alpha and beta prefoldin in soluble and insoluble forms. A) αPFD-SpyCatcher (MW: 29.61399 kDa) B) βPFD-SnoopCatcher (MW: 28.229 KDa)
A: αPFD-SpyCatcher
B: βPFD-SnoopCatcher.
Purification
Following confirmation from protein expression assay (Figure 2), αPFD-SpyCatcher and βPFD-SnoopCatcher were purified by immobilised metal affinity chromatography (IMAC) using a Ni-NTA column connected to an AKTA Start chromatography system. Elution fractions 3-11 were collected and run on SDS-PAGE (Figure 2).
Figure 3. SDS-PAGE of AKTA purification fractions (F3-9 and F11). A) αPFD-SpyCatcher (MW: 29.61399 kDa) B) βPFD-SnoopCatcher (MW: 28.229 KDa).
A: αPFD-SpyCatcher
B: βPFD-SnoopCatcher.
Assembly
Assembly of the hexameric Assemblase scaffold was visualized using a Native-PAGE gel (Figure 3). The Assemblase scaffold (2 αPFD-SpyCatcher/4 βPFD-SnoopTag) can be seen to increase in concentration as more βPFD-SnoopCatcher is added to the αPFD-SpyCatcher, while αPFD-SpyCatcher decreases in concentration as increasing amounts of βPFD-SnoopCatcher is added.
Figure 3. Native-PAGE gel showing assembly of alpha and beta subunits into the hexameric Assemblase structure by adding βPFD-SnoopCatcher to αPFD-SpyCatcher at different molar ratios.
Conjugation
To show the function of the Spy- and SnoopCatcher-Tag system, the fluorescent proteins, mVenus-SpyTag and mCerulean3-SnoopTag, were conjugated onto αPFD-SpyCatcher and βPFD-SnoopCatcher, respectively (Figure 4).
Figure 5. SDS-PAGE showing conjugation of fluorescent proteins to prefoldin (PFD). A) αPFD-SpyCatcher conjugated with mVenus-SpyTag. B) βPFD-SnoopCatcher conjugated with mCerulean3-SnoopTag.
FRET
A Förster resonance energy transfer (FRET) experiment was used to demonstrate that successful scaffolding of two different proteins to the α/β-PFD scaffold, as well as the self-assembly of the scaffold with proteins attached led to functional communication between the attached proteins. Purified mCerulean3-SnoopTag and mVenus-SpyTag were conjugated to βPFD-SnoopCatcher and αPFD-SpyCatcher (respectively) to form the complex for FRET. Unfortunately, however, no FRET signal was detected (Figure 5).
Figure 6. Emission spectrum from the FRET experiment run on assembled scaffold conjugated to fluorescent proteins (mCerulean3-SnoopT and mVenus-SpyT) as well as the fluorescent proteins without conjugation to the scaffold. The emission maxima for mCerulean3 and mVenus are 475 and 528nm respectively1.
Assembly was tested using two methods: i. Assembly of the prefoldin scaffold prior to the addition of fluorescent proteins, and ii. The conjugation of fluorescent proteins to their respective prefoldin prior to scaffold assembly. However, neither method showed any FRET signal. SDS-PAGE was run on the samples to visualize the conjugation of fluorescent proteins to the prefoldin scaffold (Figure 6).
Figure 7. SDS-PAGE gel of samples tested using FRET analysis.
A: Assembly of the scaffold performed prior to conjugation of fluorescent proteins.
B: Tag/Catcher conjugation of proteins performed prior to scaffold assembly.
Discussion
A protein expression assay was used prior to purification in order to determine protein solubility. The BugBuster gel in Figure 1A shows αPFD-SpyCatcher as soluble while Figure 1B shows βPFD-SnoopCatcher in the insoluble fraction. However, during purification, βPFD-SnoopCatcher appeared soluble.
Following large scale grow up, the cells were induced at an OD600 of 0.6 with 1mM IPTG and incubated overnight at 20°C. Cells were pelleted and lysed by sonication, and the soluble fraction was purified via immobilised metal affinity chromatography using a Ni-NTA column connected to an AKTA start chromatography system. Elution fractions thought to contain the protein (determined by observing the AKTA start chromatogram) were run on SDS-PAGE gel (Figure 2). A large amount of contamination from unwanted proteins were seen in the early elution fractions. Fractions 9-11 for αPFD-SpyCatcher and fraction 11 for βPFD-SnoopCatcher had little contaminants and were thus concentrated and buffer exchanged using an Amicon Ultra 15 ml, 10 KD concentrating column. Afterwards, protein concentration was determined using a BCA assay. This protein preparation could then be used for further experiments as described below.
In order to show the formation of Assemblase, constant amounts of αPFD-SpyCatcher were incubated with increasing quantities βPFD-SnoopCatcher for approximately 30 hours at 4°C. These samples were then run on a native-PAGE gel (Figure 3) to not disrupt the hydrophobic interaction (via β-barrel formation) between α and β prefoldin subunits. Assemblase is seen to increase in concentration as more βPFD-SnoopCatcher is added. This demonstrates the ability of our scaffold to self-assemble.
To establish the ability of our scaffold to conjugate proteins via the Spy- and Snoop Catcher/Tag system, the fluorescent proteins: mVenus-SpyTag and mCerulean3-SnoopTag were expressed and purified. These were then added to αPFD-SpyCatcher and βPFD-SnoopCatcher respectively in different ratios and run on SDS-PAGE gel (Figure 4). Good conjugation is seen with a dark band observed at approximately 60 kD (the combined molecular weight of prefoldin and fluorescent protein).
These recombinantly produced fluorescent proteins form a FRET pair, mCerulean3 as the FRET donor and mVenus as the FRET acceptor. These fluorescent proteins were conjugated to their respective prefoldins and combined to form the full hexameric structure. A FRET experiment was performed on the assembled structure (with increasing concentrations of scaffold) as well as on appropriate controls, however no FRET was achieved as indicated in Figure 5. This was confirmed by comparing the emission spectrum of scaffold with proteins with the spectra of controls including both fluorescent proteins (no scaffold), donor protein only, acceptor protein only, donor, acceptor and αPFD-SpyCatcher and donor, acceptor and βPFD-SnoopCatcher. If the two proteins are brought close together via the scaffold and mCerulean3 is excited by a wavelength of 433 nm, its fluorescence emission (which roughly ranges from 475 to 515 nm) should excite mVenus and result in an additional fluorescence emission at 530 nm (mVenus emission), as well as a decrease in fluorescence at 475 nm (mCerulean3 quenching). This result was not achieved with no peak corresponding to mVenus emission being present in Figure 5.
Two strategies for scaffold conjugation and assembly were tested including assembling the scaffold prior to the simultaneous conjugation of mCerulean3 and mVenus via the Spy/Snoop Catcher-Tag system. The conjugation of αPFD-SpyCatcher and βPFD-SnoopCatcher to mVenus and mCerulean3 respectively prior to scaffold assembly was also tested with data shown in Figure 5. Both methods yielded similar results, with no increase in emission at 530nm relative to the emission at 475nm being observed.
Following this result, the samples were run on an SDS-PAGE gel (Figure 6). Although the SDS-PAGE cannot show the full scaffold of Assemblase as all non-covalent bonds are denatured, any conjugation between fluorescent protein and prefoldin can be visualized. Figure 6 shows limited conjugation indicating a significant amount of free fluorescent proteins are present, which is likely to be the reason for no FRET signal. This result is unexpected as assembly and conjugation gels in Figures 3 and 4 indicate successful assembly between βPFD-SnoopCatcher and αPFD-SpyCatcher, as well as successful conjugation between fluorescent protein and prefoldin. Low conjugation observed in these FRET experiments may have occurred due to inadequate storage conditions of the fluorescent protein and prefoldin stock solutions.
Due to time constraints, further tests could not be performed. However, in an effort to demonstrate complete assembly by FRET, future tests would include re-expression and purification of each protein. The quality of the assembly would then be verified on Native-PAGE and additional conjugation experiments would be performed in order to ensure appropriate ratios of scaffold to fluorescent proteins. Storage conditions should always be monitored with the proteins kept at 4°C and these experiments should be performed within as little time as possible to avoid inactivation or precipitation of proteins, which may explain the inefficient Tag/Catcher conjugation observed towards the end of our project. Moreover, the complete assembly could be separated from excess prefoldin scaffold and/or free fluorescent proteins using size exclusion chromatography or multimodal chromatography. The pure hexameric, functional scaffold could then be used to perform FRET experiments and we would likely have more success due to no excess free fluorescent proteins (incapable of the FRET effect) being present in the reaction.
References
- Jonáš, A. et al. In vitro and in vivo biolasing of fluorescent proteins suspended in liquid microdroplet cavities. Lab Chip 14 3093-3100 (2014).