A big part of iGEM is creating something useful for future iGEM teams. Read about what we did to achieve this.
Expressing proteins in plants has been done by former iGEM teams, but is still a rarity, which is in accord with the industry. Therefore, here lies a great potential not only for future teams, but also for companies. What was missing as we began working on our project was a piece of compact advice on how to approach plant expression. This is why we, additionally to providing parts necessary for in planta cyclization of different peptides, also decided to write down a short guide on plant-based protein expression for future teams interested in employing this method in their own projects.
Adding New Basic and Composite Parts
To the database we’ve added new basic parts required for cyclization of peptides in general:
- BBa_K3757000 - Asparaginyl endopeptidase 1, also known as butelase 1, possessing a ligase activity required for cyclization of peptides grafted into the Oak1 precursor.
- BBa_K3757001 - Oak1, so called “precursor protein of the cyclotide katala B1”, containing N- and C-terminal propeptides needed for ER-localization, vacuole translocation, and AEP processing of the cyclotide contained between the propeptides.
- BBa_K3757002 - McoTI-II, a squash trypsin inhibitor cyclotide with exceptional heat and protease stability, possessing no antibacterial, hemolytic or cytotoxic activity. Three disulfide bridges lead to formation of six loops. Loop number five was modified to contain His6-tag.
- BBa_K3757003 - McoTI-II, a squash trypsin inhibitor cyclotide with exceptional heat and protease stability, possessing no antibacterial, hemolytic or cytotoxic activity. Three disulfide bridges lead to formation of six loops. Loops number one and five were modified to contain CHEN and His6-tag, respectively.
- BBa_K3757004 - McoTI-II, a squash trypsin inhibitor cyclotide with exceptional heat and protease stability, possessing no antibacterial, hemolytic or cytotoxic activity. Three disulfide bridges lead to formation of six loops. Loops number six and five were modified to contain CHEN and His6-tag, respectively.
- BBa_K3757005 - McoTI-II, a squash trypsin inhibitor cyclotide with exceptional heat and protease stability, possessing no antibacterial, hemolytic or cytotoxic activity. Three disulfide bridges lead to formation of six loops. Loops number one and five were modified to contain KR-12 and His6-tag, respectively.
- BBa_K3757006 - McoTI-II, a squash trypsin inhibitor cyclotide with exceptional heat and protease stability, possessing no antibacterial, hemolytic or cytotoxic activity. Three disulfide bridges lead to formation of six loops. Loops number six and five were modified to contain KR-12 and His6-tag, respectively.
These basic parts were then used by our team to create new composite parts we’ve used in our project:
- BBa_K3757007 - composite part, gene containing Oak1 with a C-terminal myc-tag, regulated by a constitutive CaMV 35S promoter and 35S terminator polyadenylation signal. Codon-usage was optimized for expression in Nicotiana benthamiana (N. benthamiana).
- BBa_K3757009 - composite part, gene containing sGFP (S65T), regulated by a constitutive CaMV 35S promoter and 35S terminator polyadenylation signal. Codon-usage was optimized for expression in N. benthamiana.
- BBa_K3757010 - composite part, gene containing CtAEP1, regulated by a constitutive CaMV 35S promoter and 35S terminator polyadenylation signal. Codon-usage was optimized for expression in N. benthamiana.
- BBa_K3757011 - composite part, a 3in1 binary vector containing Oak1 and CtAEP1 required for the cyclization process, as well as sGFP(S65T) as a form of expression control.
We are convinced, that especially our composite part BBa_K3757011 can be useful for any future iGEM teams interested in enhancing stability of their (antimicrobial) peptides of interest. As this part is contained within a binary vector used for Agrobacterium tumefaciens mediated transient expression in N. benthamiana , we have additionally created a compact guide with steps necessary for successful implementation of this method. Besides, it involves references to further resources that might prove to be useful, as well as optimalization suggestions that one might need to consider to obtain the highest possible yield of the peptide of interest. This guide can be used by teams not only interested in using our chassis to boost the stability of their peptides, but also by teams interested in plant-based expression in general.
Plant Expression Guide
The first step in ensuring successful Agrobacterium mediated protein expression in plants is choosing the right vector to carry your cloned construct. We recommend using our 3in1 vector if your team is interested in engineering more stable peptides. However, if you are interested in using a plant expression system in general, we recommend following reviews as an overview of what possibilities there are.
In particular in terms of using the right plasmids and making sure it contains all genes, signals and motifs. For example, two different origins of replication: One Escherichia coli specific, as cloning and propagation of the plasmid is conducted in this organism, as well as one A. tumefaciens specific for the successful propagation of transformed Agrobacteria .
Nevertheless, exploring other options might prove useful for your project. These involve methods of combining the Agrobacteria based approach with the CRISPR/Cas9 systems, or viral systems to achieve higher expression and yield.
Gleba Y.Y., Tusé D., Giritch A. (2013) Plant Viral Vectors for Delivery by Agrobacterium. In: Palmer K., Gleba Y. (eds) Plant Viral Vectors. Current Topics in Microbiology and Immunology, vol 375. Springer, Berlin, Heidelberg. https://doi.org/10.1007/82_2013_352
Gordon, J. E., & Christie, P. J. (2014). The Agrobacterium Ti Plasmids. Microbiol Spectr, 2(6). doi:10.1128/microbiolspec.PLAS-0010-2013
Zhang, Y., Zhang, Q., & Chen, Q. J. (2020). Agrobacterium-mediated delivery of CRISPR/Cas reagents for genome editing in plants enters an era of ternary vector systems. Sci China Life Sci, 63(10), 1491-1498. doi:10.1007/s11427-020-1685-9
Once the cloning and purification of the plasmid is finished, the transformation of Agrobacteria is necessary. Here, three different options are possible. Two are very similar to typical methods used with E. coli as well, heat-shock transformation and electroporation. If you decide to use electroporation, consider this already when purifying the plasmid as salt residues might lead to killing the bacteria in the process. The third one is called triparental mating and can be useful when the plasmid is quite large. For more details and a reliable protocol, we recommend this paper.
Wise, A. A., Liu, Z., & Binns, A. N. (2006). Three methods for the introduction of foreign DNA into Agrobacterium. Methods Mol Biol, 343, 43-53. doi:10.1385/1-59745-130-4:43
After successful transformation, grow the Agrobacteria to a sufficient amount corresponding to the scale of your experiment. This is also related to the method of infiltration being used, as using syringes doesn’t require as much material, when compared to spraying or vacuum infiltration. To ensure the highest transformation efficiency possible, we also recommend mixing your strain of Agrobacteria 1:1 with Agrobacteria carrying the p19 construct, as this helps to suppress plants innate immune response. In addition, should your team be interested in creating stable lines instead, we suggest to read more on a method call “floral dip” to create transgenic lines.
Stephenson, M. J., Reed, J., Brouwer, B., & Osbourn, A. (2018). Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale. J Vis Exp(138). doi:10.3791/58169
Voinnet, O., Rivas, S., Mestre, P., & Baulcombe, D. (2003). An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J, 33(5), 949-956. doi:10.1046/j.1365-313x.2003.01676.x
Zhang, X., Henriques, R., Lin, S. S., Niu, Q. W., & Chua, N. H. (2006). Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc, 1(2), 641-646. doi:10.1038/nprot.2006.97
Three to five days post infiltration of N. benthamiana leaves, the production of the protein should peak. To make sure whether everything worked as expected, building an expression control into your plasmid is necessary. We highly recommend using fluorescent proteins such as GFP, as these enable fast screening for successfully transformed plants with a simple epifluorescence microscope. However, as not all laboratories do have one in possession, other alternatives, such as β-glucoronidase (a glycosidase, whose activity can be monitored via an assay) are available.
In the next step, harvesting of the leaves can be performed by cutting them of the remaining plant. However, this is suitable for small scale applications only. Once you are interested in big scale manufacturing, whole plants can be harvested as time/yield trade-off is not significant at this point anymore. The extraction of your desired proteins from the leaves can be performed manually by pestling under liquid nitrogen. But again, for large scale applications, it is recommended to use different methods instead, for instance ribolysing or a juicer.
Conlon, H. E., & Salter, M. G. (2007). Plant Protein Extraction. In E. Rosato (Ed.), Circadian Rhythms: Methods and Protocols (pp. 379–383). Humana Press. https://doi.org/10.1007/978-1-59745-257-1_28
Giritch, Anatoli; Marillonnet, Sylvestre; Engler, Carola; van Eldik, Gerben; Botterman, Johan; Klimyuk, Victor; Gleba, Yuri (2006): Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors. In: Proceedings of the National Academy of Sciences 103 (40), S. 14701–14706. DOI: 10.1073/pnas.0606631103.
Poon, S., Harris, K. S., Jackson, M. A., McCorkelle, O. C., Gilding, E. K., Durek, T., van der Weerden, N. L., Craik, D. J., & Anderson, M. A. (2018). Co-expression of a cyclizing asparaginyl endopeptidase enables efficient production of cyclic peptides in planta. Journal of Experimental Botany, 69(3), 633–641. https://doi.org/10.1093/jxb/erx422
Potula, H. H. Surya Kumar; Kathuria, Sonal Roy; Ghosh, A. K.; Maiti, T. K.; Dey, S. (2008): Transient expression, purification and characterization of bioactive human fibroblast growth factor 8b in tobacco plants. In: Transgenic research 17 (1), S. 19–32. DOI: 10.1007/s11248-007-9072-4.
After extraction, purification is the last step before obtaining your protein of interest. Here, a lot is again dependable on the protein itself, but generally, any tag-based method can be employed. If you are however interested in native proteins without a tag, HPLC will be your method of choice.
We hope, that this short guide inspires you and helps you on your way of using plants as bioreactors for manufacturing your favorite peptide or protein.
Pros and Cons of Different Techniques
Pros and Cons Syringe
takes a long time
Pros and Cons Vacuum
can be used to upscale the process
needs a lot of agrobacteria
Pros and Cons Spraying
can be used for big fields, doesn't need a lot of agrobacteria
Pros and Cons Pestling
(Cooling the plant material with liquid nitrogen and pestling it per hand)
elaborated process, liquid nitrogen is expensive
Pros and Cons Ribolyser
(a machine which destroys cells through fast-moving glass particles)
not as effective
Pros and Cons Juicer
(a machine which destroys the cells through force mechanically)
good for large amount
not as good of an extraction
Aqueous Buffer (e.g., HEPES)
usable for His-tag purification
probably too weak to extract proteins from plant material
Pros and Cons Lämmli
As first, fast, extracts everything
can´t be used for purification
Pros and Cons Acetonitrile
can also be used for hydrophobic proteins
denatures and degrades Proteins, needs an organic solution
Pros and Cons Tag
very specific, good for big proteins, easy in the technical aspect, can be detected through antibodies
not usable for small proteins, don´t know if it changes the structure of the protein
Pros and Cons HPLC
no tag needed, good for small proteins
not very specific