The current industry practices for chlorophyll removal from green seed canola oil involve the use of acid-activated clays (AACs). However, AACs are associated with a variety of problems, including product loss, lack of reusability, and environmental concerns.
The molecular structure of AACs causes them to bind to chlorophyll and retain large volumes of oil, resulting in a 20% loss of oil during processing. The irreversible binding between the AACs and chlorophyll prevents reuse of the AACs, leading to increased costs for the manufacturer. This also increases the amount of waste produced by the system. The waste is mixed into animal feed and released into the ecosystem, where it has the potential to alter soil pH and nutrient availability for future crops. Our team sought to find an alternative method of oil-processing that addresses all three issues presented by AACs.
Identifying a WSCP
Our literature review led us to chlorophyll binding proteins, which can be found in two forms: membrane-bound and water-soluble (Takahashi et al., 2012). During the initial stages of project design, our team explored the idea of creating a biofilm using bacteria which express a membrane-bound chlorophyll binding protein on their surface to filter chlorophyll out of oil. To gain insight into the feasibility of the use of a biofilm in oil purification, we contacted Dr. Joe Harrison,a microbiology professor at the University of Calgary who focuses on biofilms.
Dr. Harrison explained that a biofilm may not work for industrial oil purification, due to the bacteria's inability to express high amounts of surface proteins and the hydrophobicity of biofilm that would prevent the oil from filtering through. Additionally, this method would expose the bacteria directly to the oil, which would raise major food safety concerns. Lastly, we found that these membrane-bound chlorophyll binding proteins primarily incorporate chlorophyll into their structure as they embed into the membrane, which would greatly reduce their chlorophyll-capturing ability in this application.
Recognizing the drawbacks associated with using membrane-bound chlorophyll binding proteins and biofilms, we turned our attention towards water-soluble chlorophyll binding proteins (WSCPs). We spoke with Dr. Ian Lewis, a biochemistry professor at the University of Calgary, who brought to our attention another issue: the hydrophobic effect.
Because WSCPs are hydrophilic in nature, they are unlikely to retain structure and function in a hydrophobic environment such as oil (Rau et al., 2001). Dr. Lewis told us that we would need to keep our WSCPs in a hydrophilic environment while still allowing it to come in contact with the chlorophyll in the hydrophobic phase. However, he did agree that mass-producing and purifying the soluble cytoplasmic chlorophyll binding protein would allow us to maximize the protein to chlorophyll ratio, which was a prominent issue for the biofilm system.
Encouraged by this meeting, we began investigating ways in which we could maintain WSCP functionality in a hydrophobic environment. We ultimately decided to utilize an emulsion system with an aqueous phase containing the water-soluble chlorophyll binding protein that would be emulsified in oil. This system allows the protein to stay in the aqueous phase where it will remain functional, whilst maximizing the surface area of the oil-water interface where proteins and chlorophyll may come into contact with one another (Bednarczyk et al., 2015). The oil and water phases can be subsequently separated, resulting in an aqueous phase containing the WSCP-chlorophyll complex and a refined oil product.
To verify the feasibility of our proposed chlorophyll removal technique, we contacted Dr. Raymond Turner and Dr. Marie Fraser, two biochemistry professors at the University of Calgary. Dr. Turner told us to be aware of any potentially toxic contaminates that come in contact with that oil at any point in the experimental workflow, and stressed the need to purify our protein before use in the emulsion. In addition, Dr. Turner advised us to consider using signal peptides to secrete our protein, as the periplasm would have fewer potential toxic compounds. Secretion to the periplasm may also contribute to higher protein yields in some systems. In terms of the emulsion system itself, Dr. Fraser informed us that the emulsion and the protein would have to be very stable to allow the chlorophyll to move through the phases.
Figure 1. 6GIX tetramer bound to four chlorophyll molecules (shown in green)
Taking this valuable advice into account, our team began investigating different WSCPs. Multiple characterized WSCPs were found on the NCBI Protein Data Bank, but the WSCP isolated from Lepidium virginicum, which we refer to as 6GIX, was chosen for this project due to literature that indicated successful binding of chlorophyll in an emulsion system using mineral oil (Bednarczyk et al., 2015). The NCBI Protein Data Bank also provided the 3D structure of 6GIX as determined by X-ray crystallography, which was extremely useful for our modelling efforts.
Creation of Genetic Constructs
Our genetic constructs were designed with a few key considerations in mind. We needed to maximize production of the protein while minimizing the risk of inclusion body formation. In addition, we needed to ensure that the protein we produced could easily be purified.
For an initial proof-of-concept system, we sought to maximize gene expression and protein production using the well-characterized T7 inducible promoter system in E. coli BL21 (DE3). This was paired with a bidirectional terminator to minimize antisense inhibition of expression. At the translational level, a strong ribosome binding site (RBS) was selected for the circuit and double stop codons were utilized.
We investigated various affinity tags for protein purification and decided on the well-characterized 6xHistidine (6xHis) tag for its ubiquity and simplicity. Structural protein modelling informed us that the affinity tag should be fused to the N-terminus, which is more externalized than the C-terminus of 6GIX. Though molecular dynamic simulation suggested that the 6xHis tag would not interfere with 6GIX’s chlorophyll-binding function, we also included a linker sequence with a thrombin cleavage site in case removal of the 6xHis tag was required.
Figure 2. 6GIX protein with a 6xHis tag attached to the N-terminus.
High levels of protein production associated with inducible systems may lead to the formation of inclusion bodies, where the protein of interest doesn’t fold properly and results in an insoluble aggregate within the cytoplasm. Secretion of the protein of interest to the periplasm can help recombinant proteins expressed in E. coli to fold properly, as well as simplify purification protocols (Sørensen and Mortensen, 2005).
To determine if secretion of our protein would impact the efficiency of our system, we designed our genetic constructs with a signal peptide attached to the N-terminal of the chlorophyll binding protein. 6GIX has an endogenous signal peptide that localizes it to the thylakoid membrane in plant cells (Palm et al., 2018). We removed this sequence to ensure there was no interference when we added our own bacterial signal peptide. When deciding which signal peptide to use, we discovered that their function is highly dependent on the sequence of the protein they are fused to (Freudl, 2018). Therefore, we decided to test six different signal peptide sequences with 6GIX in order to compare their efficacies. The signal peptides that we used included DsbA signal peptide (BBa_K3114000), MalE signal peptide (BBa_K3114001), OmpA signal peptide (BBa_K3114002), PhoA signal peptide (BBa_K3114003), YcbK signal peptide (BBa_K3114004), and TorA signal peptide (BBa_K3114005).
The DsbA, MalE, OmpA, and PhoA signal peptides are from the Sec secretory pathway, where proteins are secreted prior to folding and the signal peptides are cleaved from the protein of interest by endogenous cellular machinery during the secretion process. As a result, they are not expected to affect the protein’s final structure or function (Natale, Brüser, & Driessen, 2008). However, proteins with YcbK and TorA signal peptides pass through the Tat pathway, and are therefore fully or partially folded before secretion (Natale, Brüser, & Driessen, 2008). Our team turned towards molecular dynamic simulation to determine how the presence of these signal peptides may affect the folding of 6GIX.
Our modelling suggested that 6GIX was stable and correctly folded when fused to YcbK signal peptide and TorA signal peptide.
In addition, we created a genetic construct for 6GIX that did not encode a signal peptide. This part was used for cytoplasmic purification of 6GIX via affinity chromatography
We decided to use Golden Gate assembly to increase the modularity of our genetic construct assembly. We needed to create a set of genetic constructs that each had a different signal peptide sequence fused to 6GIX. Each of the basic parts was therefore designed to be both Golden Gate and BioBrick RFC compatible. The fusion sites for these parts were designed based on the MoClo standard (Weber et al., 2011).
Figure 3. Genetic circuit designed for the expression of 6GIX, with interchangeable signal peptides
We converted pSB1A3 into a Golden Gate destination vector using an improved RFP flipper device based on the part BBa_K1467400. This part was improved by substituting a strong constitutive promoter for the inducible promoter, a stronger RBS, and a double terminator, in addition to codon-optimizing this sequence for high expression in E. coli. For more information, please see our parts page.
Purification of 6GIX
Using our 6GIX genetic construct that lacked a signal peptide, we were able to successfully produce and purify the 6xHis-tagged 6GIX protein using Ni-NTA affinity chromatography as demonstrated below. The purified 6GIX was used in subsequent emulsion experiments. For more, see the Protein Emulsification page.
Figure 4. SDS-PAGE gel showing whole cell lysate and Ni-NTA purification fractions for 6GIX without a signal peptide and an empty vector control. The marker used is the NEB colour protein standard. The arrow denotes correct band size of 21 kDa for the 6GIX protein.
To make sure that this band represents the correct protein, our team ran a western blot using Mouse Anti-HIS-tag mAb (MBL) for primary antibody and Goat Anti-Mouse:HRP (Jackson ImmunoResearch Laboratories) for secondary antibody. This blot further confirmed that our 6GIX protein was successfully produced!
Figure 5. Western blot of whole cell lysate and Ni-NTA purification fractions for 6GIX without a signal peptide and an empty vector control. A His-tagged positive control was also included. The marker used is the NEB colour protein standard. The antibodies used were Mouse Anti-HIS-tag mAb (MBL) for primary antibody and Goat Anti-Mouse:HRP (Jackson ImmunoResearch Laboratories) for secondary antibody.
We were also able to secrete 6GIX with the DsbA signal peptide, MalE signal peptide, and PhoA signal peptide. The construct with the MalE signal peptide showed the strongest band of the three, but the yield was still very low compared to purification from the cytoplasm. Optimizing our secretion system may be a potential avenue for improvement in the future as we move towards industrial use.
Figure 6. SDS-PAGE gel showing periplasmic purification of 6GIX with six different signal peptides, no signal peptide, and an empty vector control. The marker used is the NEB colour protein standard. The arrow denotes correct band size of 21 kDa for the 6GIX protein.
Following a successful protein purification, our team began to analyze how we may improve our construct design for use in future scale-up. One of the issues flagged in nearly every conversation with scientists and stakeholders was the scalability of our IPTG-inducible T7 promoter system. Although this system was well-characterized, widely-used, and convenient for our initial proof of concept, the high cost of IPTG makes it unfeasible for industry use. In the future, our team would like to investigate alternative inducible systems, such as a xylose-inducible promoters to produce our protein at much lower cost. In addition, our team investigated alternatives to the Ni-NTA affinity chromatography in order to accommodate individuals with nickel allergies and minimize purification costs. In the future, we may consider using other tags, such as a biotin-carboxy carrier tag, for allergen-free purification of our protein.
Bednarczyk, D., Takahashi, S., Satoh, H., & Noy, D. (2015). Assembly of water-soluble chlorophyll-binding proteins with native hydrophobic chlorophylls in water-in-oil emulsions. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1847(3), 307–313. https://doi.org/10.1016/J.BBABIO.2014.12.003
Freudl, R. (2018, March 29). Signal peptides for recombinant protein secretion in bacterial expression systems. Microbial Cell Factories. BioMed Central Ltd. https://doi.org/10.1186/s12934-018-0901-3
Natale, P., Brüser, T., & Driessen, A. J. M. (2008, September). Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane-Distinct translocases and mechanisms. Biochimica et Biophysica Acta - Biomembranes. https://doi.org/10.1016/j.bbamem.2007.07.015
Palm, D. M., Agostini, A., Averesch, V., Girr, P., Werwie, M., Takahashi, S., … Paulsen, H. (2018). Chlorophyll a / b binding-specificity in water-soluble chlorophyll protein. Nature Plants 2018 4:11, 4(11), 920. https://doi.org/10.1038/s41477-018-0273-z
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Takahashi, S., Yanai, H., Nakamaru, Y., Uchida, A., Nakayama, K., & Satoh, H. (2012). Molecular cloning, characterization and analysis of the intracellular localization of a water-soluble chl-binding protein from brussels sprouts (brassica oleracea var. gemmifera). Plant and Cell Physiology, 53(5), 879–891. https://doi.org/10.1093/pcp/pcs031
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