Our Concept
Our ultimate goal was to develop a scalable, efficient, and accessible method for producing antimicrobial peptides (AMPs) in bacteria. We believe that in doing so, we will create a system that can be used globally to produce AMPs on a large scale, so that they can be purified and used as therapeutics. We proposed the use of an encapsulin nanocompartment to mitigate AMP toxicity to the expression bacteria.
Our team demonstrated the efficacy of our system First, our expression system effectively produced an AMP known to be toxic to E. coli in E. coli using the limited resources of our lab. Second, we purified protein from our encapsulin system via heat purification and ammonium sulfate precipitation. Third, we demonstrated that the increased metabolic burden of encapsulin production did not lower the growth rate of bacteria producing both AMP and encapsulin relative to bacteria producing AMP alone. Fourth, our computational work modeled the scalability of our expression system, showing that AMP yields could be increased without compromising expression strain viability.
With these achievements, we have successfully designed an accessible, inexpensive, and scalable expression system for biosynthetic AMP production.
Objective 1: Production of AMP toxic to E. coli in E. coli
Our AMP, HBCM2, was chosen largely for the strong literature evidence for its toxicity to gram negative bacteria, including E. coli. We ordered the genetic sequence for this peptide from IDT and successfully transformed our ordered fragment into BL21 E. coli. Upon induction of protein expression, we demonstrate the production of this peptide in E. coli through the use of our encapsulin-based expression system as demonstrated by the gel below (Figure 1). The AMP is shown at the bottom of the gel.
Figure 1. SDS-PAGE gel showing protein expression of encapsulin + AMP
Objective 2: Purification of AMP and encapsulin from expression bacteria
Following our expression of HBCM2 in E. coli, we used the thermostable and solubility properties of our encapsulin to demonstrate the purification of a clean protein sample (Figure 2). Both the expression and purification of our protein were performed using the basic molecular biology laboratory techniques and equipment available to our team. These techniques are easily scalable to large batches of AMP and encapsulin. Further, we designed our system in a way that allows for the gentle release and ready separation of our AMP from shielded encapsulation.
Figure 2. SDS-PAGE gel demonstrating ammonium sulfate purification of T4GALA encapsulin monomer and AMP
Objective 3: Demonstration that increased metabolic burden of encapsulin production does not lower the growth rate of bacteria producing both AMP and encapsulin relative to bacteria producing AMP alone
Because of the considerable size of our encapsulin, our team was concerned that the increased metabolic burden of encapsulin expression could affect the productivity of expression E. coli. Our growth assay data demonstrates that this was not the case (Figure 3). The encapsulin + AMP bacteria (yellow) did not have a lower growth rate than the AMP-only bacteria (orange).
Figure 3: Growth assay average growth rates for 4 experimental groups upon induced gene expression: empty pET vector, encapsulin only, AMP only, and encapsulin + AMP. Error bars represent 95% confidence intervals.
Objective 4: Potential to Scale
Through modeling, we showed that chaining together AMPs in polymers would improve the AMP yield, demonstrating the potential to scale the system in the future. Results from mathematical modeling suggested there was a tradeoff between bacterial growth and number of AMPs in our polymer but that AMP polymers could be created with lengths of up to around 4 before net bacterial growth was reduced to zero. This suggests a possible yield improvement of 2-4x is feasible.
To determine if raising the number of AMPs in polymers would adversely affect the ability of the TEV protease enzyme to separate then back into monomers during downstream AMP purification, we performed molecular docking experiments for AMP (HBCM2) polymers of various sizes with the TEV protease enzyme (Figure 3).
Figure 3: box-and-whisker plots of binding affinities of AMP (HBCM2) polymers of different lengths (from 1 to 7) with TEV Protease. Statistical significance is demonstrated between the binding affinities of all other polymer lengths and the pentamer and heptamer. Error bars represent standard deviation.
Results from these experiments suggested that increasing AMP polymer size has minimal adverse effects on binding affinity of TEV protease + AMP polymer. In fact, in some cases, binding affinity improved. This indicates that increasing the yield of our system through AMP polymers will not harm downstream purification and isolation of AMP monomers. While we did not have the time to verify this in vivo, our work in silico provides compelling reasons to believe that our AMP + encapsulin system can be effectively scaled.
In conclusion, we successfully demonstrated that we can produce an AMP in bacteria that is known to be toxic to E. coli. From there, we were able to purify the AMP from our encapsulin system. Furthermore, the growth rate of our bacteria was shown to be minimally affected by the production of our encapsulin and AMP compared to solely producing the AMP. Finally, our computational work showed that our system is scalable. These taken together show that the theory behind our project is solid and this system could be brought to an industrial scale. Moving forward, testing the capabilities of our system to produce the AMP chains that we modeled would allow us to further summon the feasibility of our project.