Introduction

Protein based therapeutics have been approved for clinical use due to their unique structural and functional properties. With the global market revenue for recombinant proteins being valued at over $2.9 billion USD in 2022 with a projected increase to $5.4 billion USD by 20271. there is an ever prevalent demand for the production of biologics and clinically relevant therapeutic proteins. With proteins like antibodies being used in a range of industries starting from basic immunohistochemistry to drug testing in clinical trials, there is a need to not only increase the production of such biologics but to also do so in a sustainable manner.

Imperative components of biomanufacturing include in silico protein production efficiency, energy usage efficiency, and purification efficiency. In vivo protein production activates innate cellular and antibody-mediated cytotoxicity to avoid toxic byproduct build up in cell systems2. This creates a bottleneck for proteins that are difficult to produce, such as membrane proteins, cytokines, antibodies and many more. We aim to bypass this bottleneck through PILOT, which uses a non-living system called cell free protein synthesis.

Orthogonality

The orthogonal or perpendicular engineering approach to synthetic biology has be come quite popular in recent decades. Orthogonality in genetic circuits refers to the inability of two or more biomolecules to interact with each other or affect their respective substrates. These biomolecules are likely similar both in terms of structure and composition3. Our system's orthogonality addresses three essential pillars: energy production, protein synthesis, and protein purification. PILOT's modular design allows users to meticulously tailor different aspects of their projects according to specific requirements or goals. This segmentation liberates PILOT from the constraints of conventional CFPS systems, facilitating expedited production, scalability, and streamlining overall efficiency.

By using CFPS to create a modular platform, our team aimed to develop a high efficiency protein production system that utilises cheap substrates and allows for simultaneous protein biosynthesis, extraction and purification.

Protein Production Efficiency

Energy Usage
Efficiency

Protein Purification Efficiency

Employing rapid dividing Vibrio natriegens as a chassis for cell free protein synthesis.

Using oxidative phosphorylation instead of substrate level through split inteins.

Thiol-sensitive intein for affinity tag purification aid.

Figure 1: The three pillars of protein production P.I.L.O.T seeks to tackle.

Protein purification efficiency

With the development of modern technology, recombinant protein production has seen many advancements in protein production and purification efficiency. Isolating a protein in its active from the expression vector it is produced in can be challenging as there are many factors like proteolysis, inclusion bodies and affinity tags that need to be considered. Furthermore protein isolation depends on the nature of the protein itself i.e., whether it is soluble, insoluble, a membrane or glycoprotein. Each class has its own extensive method of purification. For example, isolating insoluble proteins demands several denaturation steps and it is highly imperative that the final purified protein is folded and oxidized correctly4. These methods of protein purification require not only extreme care but also specialised equipment such as french presses, ultrasonic homogenizers and chromatography systems.

Thus, commonly used methods of protein purification are time consuming and can be expensive. It is important to take accessibility into account especially in this situation. There are many globally who do not have access to such equipment and do not have the resources to purchase them. For this, we decided to develop a system that allowed for a one pot biosynthesis and purification reaction. By simple centrifuging, the protein of choice would be isolated into the supernatant after the reaction runs its course.

The advent of Cell Free Protein Synthesis (CFPS) platforms

When a living cell is used to produce recombinant proteins, we only have so much external control over it. The cell must divide its energy between metabolic processes to produce the desired protein and keep the cell itself alive. As the system undergoes the added stress of producing recombinant proteins in concentrations it typically has not needed to before, cytotoxicity can build up very quickly if left unchecked. Thus, there is only so much protein this system can produce before it is killed by innate defence mechanisms.

CFPS reactions are not living systems.

The use of a cell free environment to produce recombinant proteins has brought in vitro protein production a long way from what it used to be. The open environment not only allows direct manipulation but also allows for higher protein titers to be produced as the entirety of the system's energy is going towards protein production5. This allows us to therefore produce desired and labeled proteins that pose cytotoxicity threats to cells. The open environment of the reaction allows users to directly add or synthesize new ingredients at precise concentrations, allowing them to be designed, tested and optimized for different products in a faster, more convenient, and more controlled manner6.

Figure 2: Graphic demonstrating a cell free protein synthesis reaction and its components.

Lysates can be produced ahead of time, lyophilized, and stored for months, so protein production requires only the reconstitution of the lysate with CFPS reaction buffer and the addition of an expression plasmid. The simplicity of CFPS makes it suitable for use in a wide range of settings, and it does not require significant investment in specialized equipment. Whether it is the heterologous expression of membrane lysing antimicrobial peptides, or cytotoxic proteins with antitumor activity, CFPS can fulfill a continual need for protein expression for investigation or purification.

Our team aimed to take advantage of such highly efficient existing platforms and modify it such that it would possibly generate even higher protein titers by improving energy usage.

Protein Production Efficiency

Few prokaryotic platforms have the ability to produce adequate protein yields. To ensure high protein production and ease of usage, we chose Vibrio natriegens as our chassis over the conventional Escherichia coli.

During our ideation process, we came across different organisms whose lysates had been used for cell free protein synthesis. Recent studies showed the use of fast growing bacteria as potential chassis choices in CFPS preparations. Among such bacteria, the Gram-negative γ-proteobacterium Vibrio natriegens (VN) represents a promising candidate that possesses remarkably high growth and substrate consumption rates7 8 9. A natural halophile, V. nats exhibits 60% greater ribosome density relative to E. coli and doubles in half the time of E. coli10. The extremely high quantity of ribosomes from V. nats cell lysates allows high-throughput protein synthesis, especially in techniques such as ribosome display, where the limiting factor in library size is the number of ribosomes.

V. nats also has many advantages over E. coli in terms of CFPS preparation; V. nats cultures can be grown to stationary phase for optimal cell lysate quality while E. coli cell lysate must be harvested at early exponential phase, limiting lysate yields per litre of culture 11. Its halophilic nature allows it to grow comfortably in saltwater, reducing freshwater demands, while also providing the benefit of reduced media sterility requirements. It grows rapidly in minimal medium supplemented with various carbon sources under both aerobic and anaerobic conditions8. This allows a reduced reliance on extensive sterility measures that not all laboratories may have access to.

With the extensive support provided by UBC Biofoundry, we obtained a strain of Vibrio natriegens Vmax. Using this strain and plasmids under the control of a T7 promoter, protein yields are over 25-fold higher than those achieved using commercial strains and more than 10-fold higher than those achieved using E. coli12.

Energy Usage Efficiency

Current CFPS reactions can only utilise substrate level phosphorylation due to the lack of membrane associated electron transport chains; this greatly reduces energy usage efficiency to just ~5% of the total energy attainable from oxidative phosphorylation13. Inverted inner membrane vesicles (IMVs) containing respiratory proteins (where H+ is pumped into the vesicle and ATP synthase F1 subunit is on the "cytoplasmic" side) can allow oxidative phosphorylation14. IMVs are generated from lysis of the bacterial cell membrane using a homogenising process. The most important aspect of IMVs is that they contain the electron transport chain in the right orientation, allowing us to use oxidative phosphorylation and regenerate NAD from NADH.

Figure 3: Diagrammatic representation of ATP production from the electron transport chain

Oxidative phosphorylation allows use of a greater breadth of cheaper, renewable, and more energy dense fuel sources such as methanol. Full oxidation of methanol in a cellular system can generate up to 50% more ATP per carbon compared to glucose or glycerol15. Furthermore, it is a well established C1 platform chemical that can be synthesised efficiently from a variety of "waste" or cheap carbon sources such as CO2, biomass, and natural gas. Compared to other carbon sources such as succinate, glucose, glycerol or ethanol whose synthesis from CO2 is infeasible and much less efficient, methanol is an extremely energy source.

We have integrated methanol into V. nat's central metabolism in an enzyme dependent pathways by using formolase, methanol dehydrogenase (Mdh) and dihydroxyacetone kinase (DhaK). By titrating methanol into the cell lysate prior to running the CFPS, the system then utilizes and metabolizes methanol for protein production.

Where P.I.L.O.T comes in:

By using Vibrio natriegens as a chassis and employing IMVs, we have designed a system using genetic blocks called inteins, natural splicing tools in living systems . We have used two types of inteins that directly impact two of our biomanufacturing pillars namely protein purification and energy usage. Protein purification is improved by the use of a Mxe GyrA intein16 and energy usage efficiency is built in using the Nrdj-1 split intein17 system in a dual plasmid manner.

Inteins

A protein's function does not begin immediately after translation. Cells must ensure it is folded in the right configuration while making sure it is appropriately ordered or disordered based on the structure and function. Auto processing domains are classes of proteins used to modify peptide backbones and comprise of a conserved family of proteins called inteins18.

They are natural self cleaving post translational modifications endogenous to a subset of all domains of life19. These self-splicing polypeptides can excise themselves from flanking host protein regions thus ligating flanked host protein fragments. Intein-based purification of protein products streamlines the process, allows synthesis of cyclic peptides (important in AMPs), and provides flexible intermediates that can undergo a number of useful modifications.

Figure 4: Schematic of intein splicing mechanism. Adapted from Shah & Muir, 2014.

Intein mediated splicing is highly spontaneous and requires no external energy sources. Relative to simple protein-tag fusions, protein-intein-tag fusions offer the advantage of traceless purification, where no remains of the tag or intein remain after the purification process. In addition, protein-intein-tag fusions can be recovered with greater efficiency than protein-tag fusions20.

Genetic Circuits

Three major constructs went into our design consideration to validate the modularity of our system. Each was equipped with a fluorescent reporter to help quantify RFU values upon expression in a cell free system thereby helping partially confirm protein folding and production. All constructs were under the control of a T7 promoter, as the T7 RNA polymerase is inducible with IPTG. The T7 polymerase binds to the T7 promoter on our expression vector to begin transcribing the gene of interest, in our case the fluorescent reporters StayGold and mCerulean. IPTG mimics the structure of allolactose and thus binds to the lac repressor thereby preventing from inhibiting reporter gene expression. It is well studied and shown that expression of recombinant proteins under the control of this promoter is 8 times faster than protein expression under the control of the E. coli RNA polymerase21.

Expression Vector

Figure 5: Circuit diagram of expression vector. The cut sites enable the GOI to be swapped out for any protein of interest.

The design of the expression vector is directed towards the protein purification pillar of our project. Designed with a Mxe GyrA intein-ELK16 system, this construct allows for traceless purification of any protein of interest. The ELK16 tag self-assembles into an insoluble protein aggregate, allowing us to easily isolate our recombinant protein of interest by simple centrifugation22. Afterwards, adding catalytic amounts of any thiol then activates the self-splicing Mxe GyrA intein, and releases the pure protein of interest into solution which is then restored by simple cenetrifuging

Mxe GyrA Intein

The Mycobacterium xenopi GyrA (Mxe GyrA) intein is a 198 amino acid long sequence characterized in 1997. It is the smallest known naturally occurring active intein that has lost residues required for endonuclease activity. Due to the nature of its small size, it colloqially represents the most minimal structure needed for post translational protein modification23.

Figure 6: PyMol rendered model of the MxeGyrA intein. PDB file obtained from https://www.rcsb.org/structure/1am2.

In our design, site directed mutagenesis creates a "T3C" mutant of the Mxe GyrA intein in which a threonine adjacent to the primary catalytic residue (Cys-1) is converted to a cysteine. This T3C mutant results in the formation of a disulfide bond between the catalytic cysteine and neighbouring cysteine. This inactivates the intein under oxidizing conditions, preventing premature cleavage of the tagged protein. Upon reduction of the disulfide bond by thiols usually used to facilitate target protein release from the Mxe GyrA intein, catalytic activity is restored. This is why addition of minute amounts of thiol activates the seelf splicing activity of the intein to release pure protein.

Toolkit Modularity

The modularity and orthogonality of the toolkit arises from this assembly as BsaI and BamHI enzyme cut sites surround the gene after the promoter and before the intein. The gene can thus be swapped out for any sequence of interest to produce the desired protein. We originally planned to validate our expression vector through the production and traceless purification of Staygold, a green fluorescent protein which was chosen due to its extremely bright fluorescence (three times brighter than sfGFP), fast folding time (equivalent to the superfolder mNeonGreen), and exceptional photostability24.

Figure 7: PyMol rendered model of the MxeGyrA intein fused with StayGold. PDB file.
Figure 8: Circuit diagram of expression vector with proof of concept IL-10.

After we confirm the system works via Staygold fluorescence, the enzyme cut sites give us the flexibility to replace the reporter protein with clinically or industrially relevant protein of interest, including cytokines such as IL10. This plug and play format is the fundamental basis in building a modular expression program for use by anyone.

IL-10 was selected as the next step towards achieving our proof of concept goal due to its ideal use in the clinic as a potential candidate for immune modulatory effects when engineered for more fine tuned interactions with other immune cells (Gorby et al., 2020). IL-10 is one of many proof of concept biologics that can be implemented in PILOT's bioproduction platform25.

Adopting PILOT in Multi-Domain Protein Synthesis

While PILOT's concept appears to be amenable for a variety of small molecules and proteins, our hope is to expand PILOT for application to larger multi-domain protein families. Among these are the recombinant antibody class - especially important immunotherapies. We recognize that these are multi-domain, relatively large proteins whose subunits fold independently of themselves. They are difficult to chemically induce proper folding of, and require precise tertiary stabilising disulfide bonds without having to purify the antibodies and refold under proper assembly of heavy and light chain fragments if synthesised in vitro. Literature has previously established that antibodies are more precisely synthesised and correctly folded in-vivo26

Conceptually, PILOT would be able to tackle the production and resource cost of synthesising the heavy, light, or variable chains of an antibody linked together on the expression vector. PILOT would purify the chains out of solution and enable further denaturation, refolding, and reducing processes to occur for full antibody assembly. Farràs et al. tested this very concept of in-vitro Trastuzumab synthesis against in-vivo. They found that it could be refolded in vitro if the heavy and light chains were not physically separated. When done on the chains independently, the heavy chain precipitated and the original antibody structure would not be recovered, suggesting that the heavy chains require some form of interaction with the light chain to generate proper dimers..

These are important considerations for our team to include to inform future users of the PILOT toolkit about where PILOT can best help expedite selective processes of the development pipeline of more specialised biologics with complex multi-domain compositions. We are optimistic about our future development of PILOT, and pushing the frontiers of complex bio-designed molecules that it can synthesise.

Methanol Utilization Vector

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Figure 9: Circuit diagram of methanol utilization vector. Top: Part I of the vector encoding VPHVG protein condensate, one half of the Nrdj intein and mCherry and the last enzyme in the methanol pathway. Bottom: Part II of the vector encodes the first 2 enzymes of the pathway and the other halves of the Nrdj intein and mCherry

Our methanol utilisation vector employs a dual plasmid circuitry system each connected to one half of the red fluorescence protein mCherry. Our two constructs, Mdh+Fls and PC+Dhak, were each designed with half of mCherry and half of the split NrdJ intein. Upon adding both recombinant proteins to the same lysate, the split NrdJ intein would bind and splice itself out, restoring red fluorescence via the rejoined halves of mCherry. This red fluorescence would then indicate that the lysate contains all the enzymes required for methanol utilisation.

Figure 10: Overview of the methanol metabolism pathway employed by our circuits. Adapted from Zhang et al., 2018

Our Mdh-Fls construct contains the enzymes methanol dehydrogenase and formalase, whereas our PC+Dhak construct contains the enzyme dihydroxyacetone kinase (DhaK). All three enzymes are needed for the pathway to convert methanol into a usable energy source for oxidative phosphorylation. Therefore, the protein products of both the Mdh-Fls and PC-Dhak constructs need to be in the same lysate to create a high efficiency CFPS system with methanol.

NrdJ-1 Split Intein

A small fraction of the large variety of inteins found in nature encode split inteins. They are highly orthogonal, facilitating specific multi-polypeptide assemblies27. The N and C terminal of each intein is fused to one extein. Upon translation, the two intein fragments splice and assemble into the intein structure. Binding of the two domains restores intein catalytic activity, splicing the attached proteins together.

Figure 11: Schematic of split intein mechanism using mCherry. Adapted from Shah & Muir, 2014.

The Nrdj-1 split intein system is part of class II of the nine fractured loci of ribonucleotide reductase catalytic subunits. The family of loci are house keeping enzymes involved in DNA synthesis and repair28. It has an extremely high splicing rate, almost 7 fold higher than traditional DnaE and also has optimal functionality at 37° C17, closest to physiological temperatures. Each half of the NrdJ-1 split intein is attached to half of the mCherry red fluorescent reporter. The dual plasmid system operates such that methanol addition induces splicing of each half of the NrdJ system causes correct re-folding of the mCherry reporter and upon the combination of the two halves, fluorescence is restored to the solution.

Figure 12: PyMol rendered model of the Docked Nrdj mCherry. PDB file.

Successful fluorescence indicates that the system has the ability to metabolize methanol and thereby use it as an efficient carbon source.

Harnessing Dual Functionality of a Phase Separated Polymer

Purification of our expressed proof of concept protein vector in the same cell lysate as our methanol utilisation module allows for the immediate opportunity to purify proteins via temperature control. The "linked" nature of PILOT is illustrated here with the creative use of genetic elements encoding polymers that can undergo liquid-liquid phase separation - or compartmentalization of proteins and materials as a result of environmental determinants. This is commonly observed in compact cellular membraneless stress granules induced by host viral infection29.We can link the biosynthesis of our protein in our expression module to that of purification using temperature mediated phase separation strategies endogenous to cells.

The VPHVG 40x protein condensate repeat sequence was incorporated into this utilisation vector to aggregate the expressed protein and confer aggregation. This is a genetic element encoding phase behaviour of protein polymers. In an experimental study from Quiroz & Chilkoti (2015)30, VPHVG 40x was their most minimal repeat sequence exhibiting a lower critical solution temperature (LCST) near 32 degrees Celsius (near physiological 37 degrees Celsius temperature). Raising the temperature of the lysate above the LCST will disallow miscibility of the synthesised protein in the cell lysate, concentrating the protein mass as a result. Simply allowing a PILOT user to change the temperature to phase separate synthesised proteins out of the CFPS reaction allows for ease of biologics developability.

The second function of VPHVG 40x is the ability to concentrate correctly assembled split mCherry subunits to confer maximal fluorescence when light is absorbed at its absorption spectra. This a preliminary validation strategy employed by our team to ensure indirectly that mCherry is assembling as a result of the split inteins functioning as designed. We hope that more foundational advance strategies such as these will be adopted in emerging biomanufacturing platforms.

Supplementary

Plasmids from Benchling

  1. Expression vector

Figure 1a: StayGold-T3C plasmid construction from Benchling

The T3C denotes that the third amino acid of the Mxe GyrA intein has been substituted from threonine (in the original sequence from NEB's pTXB1) to cysteine, to avoid premature in vitro cleavage of the intein. The first amino acid of the intein (Cys) forms a disulfide bond with the third amino acid (Cys), preventing attack of the peptide bond until the disulfide is reduced during post-purification intein cleavage via addition of a reduced thiol. To revert the T3C construct back to the original sequence, a site directed mutagenesis was performed on the plasmid via PCR.

Figure 1b: StayGold control plasmid construction from Benchling prior to conducting site directed mutagenesis.
Figure 1c: IL-10 Proof of Concept plasmid construction from Benchling

  1. Methanol Utilization Vector

Figure 2a: Mdh+Fls construct with N terminals of NrdJ-1 and mCherry.
Figure 2b: PC + DhaK construct with C terminals of NrdJ-1 and mCherry.

Our final two plasmid constructs compose a dual plasmid circuit design which promotes methanol assimilation and serves as a validation system. One half of this dual system expresses a protein condensate and dihydroxyacetone kinase, the final enzyme in the methanol assimilation pathway. The other half of our system expresses the other enzymes in this pathway, methanol dehydrogenase and formalase. Each construct contains half of the NrdJ split intein and half of mCherry, which will fluoresce red upon the split intein properly binding and splicing itself out.

Footnotes

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  17. Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D., & Schmidt, S. R. (2012). Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. The Journal of biological chemistry, 287(34), 28686-28696. [https://doi.org/10.1074/jbc.M112.372680] ↩ ↩2

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