Legend
Scaffold
Alpha Prefoldin with SpyCatcher
Beta Prefoldin with SnoopCatcher
Enzymes
LXYL-P1-2 with SpyTag
DBAT with SnoopTag
TycA with SpyTag
PAM with SnoopTag
Introduction
The Assemblase scaffold is a fusion protein between the archaeal molecular chaperonin with SpyCatcher and Snoopcatcher, which allows for the attachment of 2 enzymes next to each other in a pathway especially if they control the rate-limiting step.
Results
Protein expression assay
Cells containing a plasmid with the αPFD-SpyCatcher or βPFD-SnoopCatcher insert (as confirmed by colony PCR and sequencing) were grown up in a large-scale overnight culture containing 1mM IPTG. A sample of this was used to perform a protein expression assay. Bug buster (a cell-lysis detergent) was then used to separate soluble and insoluble proteins.
Figure 1. Protein expression assay using bug buster to determine expression of alpha and beta prefoldin in soluble and insoluble forms. A: αPFD-SpyCatcher B: βPFD-SnoopCatcher
Purification
Following the confirmation of protein expression by bug buster gels in Figure 1, αPFD-SpyCatcher and βPFD-SnoopCatcher were purified using the AKTA start machine via a Ni-NTA column.
Figure 2. SDS-PAGE of AKTA purification fractions (F3-9 and F11). A: αPFD-SpyCatcher B: βPFD-SnoopCatcher
Assembly
Assembly of the hexameric Assemblase scaffold was visualized using a Native-PAGE gel as shown in Figure 3 below. 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
Conjugation
To show the function of the Spy and SnoopTag/catcher system, the fluorescent proteins, mVenus-SpyTag and mCerulean3- SnoopTag, were conjugated onto αPFD-SpyCatcher and βPFD-SnoopCatcher, respectively.
Figure 4. SDS-PAGE showing conjugation of fluorescent protein to prefoldin (PFD). A: αPFD-SpyCatcher conjugated with mVenus-SpyTag. B: βPFD-SnoopCatcher conjugated with mCerulean-SnoopTag.
FRET
We decided to use Förster resonance energy transfer (FRET) to demonstrate successful scaffolding of two different proteins to the α/β-PFD scaffold, as well as the self-assembly of the scaffold with proteins attached. Purified mCerulean3-SnoopTag and mVenus-SpyTag was conjugated to βPFD-SnoopCatcher and αPFD-SpyCatcher (respectively) in one tube to form the complex for FRET. Unfortunately, however, no FRET signal was detected as shown in Figure 5 below.
Figure 5. Emission spectrum from the FRET experiment run on assembled scaffold conjugated to fluorescent proteins and the fluorescent proteins by themselves
Initial assembly of the prefoldin scaffold before the addition of fluorescent proteins, as well as the conjugation of fluorescent proteins to their respective prefoldin prior to scaffold assembly were tested, however neither method showed any FRET. SDS-PAGE was run on the samples to visualize the conjugation of fluorescent proteins to the prefoldin scaffold.
Figure 6. 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 bug buster 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. It is likely to have appeared insoluble in the bug buster protein expression assay due to the pH not being at pH8.
Following large scale grow up, the cells were induced with 1mM IPTG, pelleted and lysed by sonication, and the soluble fraction was purified on a Nickel NTA column via the AKTA start machine. Elution fractions thought to contain the protein (determined by observing the AKTA start chromatogram) were run on SDS-PAGE gel as in Figure 2. A large amount of contamination from unwanted proteins were seen in the early eluted fractions, however 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 15ml, 10KD concentrating column. Afterwards, protein concentration was determined using a BCA assay. This protein stock could then be used for further experiments as described below.
In order to show the formation of Assemblase, βPFD-SnoopCatcher was added to αPFD-SpyCatcher at an increasing concentration as the structure follows a ratio of 4 β prefoldins to two α prefoldins. These samples were run on a native page gel (shown in Figure 3) to allow the formation of the double β-barrelled bond formed between α and β prefoldin subunite. 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 mCerulean-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 in Figure 4. Good conjugation is seen with s dark band observed at approximately 60kD (the molecular weight of prefoldin and fluorescent protein combined).
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. FRET was performed on the assembled structure as well as on appropriate controls, however no FRET was achieved as indicated in Figure 5. 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 (shown in 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, functionalised 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.