Difference between revisions of "Part:BBa K4143340"

Revision as of 21:48, 9 October 2022

AMP Encapsulin Composite

This composite part includes an antimicrobial peptide (HBCM2), TEV protease site, targeting peptide, and T4GALA encapsulin. The encapsulin is a protein nanocompartment that sequesters cargo carrying a targeting peptide. In this case, the antimicrobial peptide (AMP) is the cargo and it's attached to the targeting peptide to be sequestered within the encapsulin. This serves to protect the bacterial host from the AMP that it's producing [1]. The TEV protease site attaches the targeting peptide and AMP so the AMP can be isolated later on in downstream purification. This was our primary experimental construct that was compared against two controls: one with just the AMP (positive control) and one with just the encapsulin (negative control). We evaluated the encapsulin’s ability to sequester the AMP through several in-lab assays and mathematical modeling.

Figure 1: Experimental construct

Usage and Biology

Mathematical Modeling of Encapsulin + AMP

To capture the kinetics of antimicrobial peptide and encapsulin expression, AMP encapsulation, and encapsulin self-assembly, we created a model of differential equations. In this model, the encapsulin monomer (E) and AMP (P) are transcribed and translated in tandem at rates proportional to their residue length. The encapsulin monomer and AMP then form a dimer as a consequence of the targeting peptide on the AMP [1]. The pairs then assemble into the final fully-assembled encapsulin complex (C).

Figure 2: Mathematical model of encapsulin + AMP expression + encapsulation

This resulted in the following graphical output displaying AMP, encapsulin, and encapsulin complexes over time (Figure 3).

Figure 3: Predicted kinetics of AMP and encapsulin production and self-assembly

Here, we see the encapsulin monomer and AMPs quickly reach steady state levels. The AMP-encapsulin monomers then slowly rise in number before plateauing. Simultaneously, the fully-assembled encapsulin complex rises linearly and never reaches a steady state value. Based on these results, the most important factor in mitigating AMP toxicity would be the targeting peptide’s efficiency.

Growth Assay

We reasoned that if the encapsulin was mitigating the toxicity of the AMP during expression, the growth rate of bacteria expressing the AMP + encapsulin would be higher than the growth rate of bacteria only expressing the AMP alone. To test this, we designed an assay in which we inoculated wells of Luria-Bertani (LB) broth containing 100 μM ampicillin with each of our four bacterial strains. Each strain featured three biological and three technical replicates. Protein expression was induced in half of the samples with 100 μM IPTG at time=0. OD600 measurements were taken by a plate reader every 5 minutes for 12 hours. Logistic regressions were performed on the growth phase of each well, and the growth rate was recorded for each. To compare these growth rates, t-tests were performed with considerations made for the variance due to our biological and technical replicates [1].

Figure 4: Growth assay average growth rates for 4 experimental groups: empty pET vector, encapsulin only, AMP only, and encapsulin + AMP (this part). Error bars represent 95% confidence intervals. A) Growth rates for uninduced gene expression B) Growth rates for induced gene expression via IPTG.

As seen in Figure 4, bacteria expressing our AMP and encapsulin had a higher average growth rate than bacteria expressing the AMP without encapsulin, suggesting the mitigating effects of the encapsulin on AMP toxicity. Although this trend was not statistically significant in our small-scale experiment, it was observed in spite of the added metabolic burden of encapsulin expression. This suggests that even if the encapsulin failed to increase the growth rate of the AMP + encapsulin bacteria relative to those expressing just AMP, the encapsulin may still have mitigated toxicity and potentially improved protein yield. Further experiments are warranted to investigate this effect, such as increasing the number of replicates or the amount of protein expressed by each cell. Increased expression could be achieved by growing the bacteria in a richer media like terrific broth (TB) media or waiting to induce with IPTG at a higher OD600 following initial bacterial growth.

Purification of Encapsulin + AMP

In parallel with the growth assay experiments, we explored the use of our encapsulin system to express and purify HBCM2. BL21 E coli were transformed with either a pETDuet+encapsulin plasmid or a pETDuet+encapsulin+AMP plasmid. These transformants were then grown at 37 degrees Celsius and shook at 250 rpm in LB broth with 100 uM ampicillin. At an OD600 of 0.6, the bacteria were induced with 200 μM IPTG to promote protein expression. The proteins were expressed for 4 hours at 37 degrees C while shaking at 250 rpm. Harvested E. coli were then lysed and centrifuged to produce a clear lysate. The thermostability of our encapsulin allowed us to then heat the clear lysate to 85 degrees C, inducing precipitation of native E. coli proteins. The efficacy of these techniques was confirmed with a protein gel (Figure 4).

Figure 5: SDS-PAGE gel showing protein purification. Note lanes 4, 6, and 7 from left showing the increasing purity of samples heated to 25, 65, and 85 degrees, respectively. From left, ladder, clear lysate using dilute lysozyme, clear lysate using moderate lysozyme, clear lysate using high lysozyme, clear lysate with higher concentration of cells per unit lysis buffer, clear lysate heated to 65 degrees, clear lysate heated to 85 degrees.

While this process produced a relatively pure protein sample, we investigated the use of ammonium sulfate precipitation for further purification. Samples of heat-purified protein were incubated at 4 degrees C for 1 hour with ammonium sulfate at concentrations ranging from 10% to 100% saturation. After resuspension of collected pellets of precipitated protein and subsequent dialysis, a protein gel showed the ability of this process to increase the purity of our protein samples (Figure 6). We found that low concentrations of ammonium sulfate were able to selectively precipitate our encapsulin-AMP complex, as seen by the distinct T4GALA Monomer and AMP fusion bands at a 10% ammonium sulfate concentration.

Figure 6. SDS-PAGE gel demonstrating ammonium sulfate purification. From left, lanes show protein pellets collected after incubating with ammonium sulfate at concentrations ranging from 10% to 100% in 10% intervals. Note the high purity of the lower concentration bands

Molecular Dynamics of AMP-Encapsulin Stability

Through molecular dynamics, we tested the stability of the interface between the AMP and the encapsulin. To make this computationally feasible, an encapsulin hexamer was used rather than the complete 240-subunit large encapsulin. The AMP shaped like a hairpin, and therefore two conformations were chosen to be tested: one with the termini facing outward (henceforth, upright), and the other with the termini facing inward (henceforth, inverted). Simulations on both systems were run for a total of 2ns, and they were analyzed using RMSD and RMSF. Both demonstrated decreased RMSD and RMSF (<1nm) when compared to the system with a single AMP in solvent, although the lack of trials and the reduced number of frames in this simulation may have been an influencing factor (Figure 7).

Figure 7: RMSD and RMSF graphs of the upright and inverted systems. Overall, systems where the AMP is in close proximity to the encapsulin wall seem to have reduced structural change over time. Note the high RMSF between residues 38 and 48.

A visualization of the AMP-encapsulin interface with the AMP highlighted is shown below (Figure 8).

Figure 8: visualization of the upright encapsulin-antimicrobial peptide complex system. Bending at the same residue as within the single-AMP system (Thr20-Thr21) are visible here, too.

Sequence and Features

References

1. Lee, T. H., Carpenter, T. S., D'haeseleer, P., Savage, D. F., & Yung, M. C. (2020). Encapsulin carrier proteins for enhanced expression of antimicrobial peptides. Biotechnology and bioengineering, 117(3), 603–613. https://doi.org/10.1002/bit.27222

2. Triggered reversible disassembly of an engineered protein nanocage Jesse A. Jones, Ajitha S. Cristie-David, Michael P. Andreas, Tobias W. Giessen bioRxiv 2021.04.19.440480; doi: https://doi.org/10.1101/2021.04.19.440480

3. Nam H, Hwang BJ, Choi DY, Shin S, Choi M. Tobacco etch virus (TEV) protease with multiple mutations to improve solubility and reduce self-cleavage exhibits enhanced enzymatic activity. FEBS Open Bio. 2020 Apr;10(4):619-626. doi: 10.1002/2211-5463.12828. Epub 2020 Mar 18. PMID: 32129006; PMCID: PMC7137792.