Transforming E. Coli with Custom Gene Fragment to Produce MP1 Cancer Fighting Peptide

Abstract:

According to Global Cancer Statistics, there were approximately 32.6 million cancer patients reported in 2015, making it one of the world’s most imminent health threats.^[1] Currently, chemotherapy is the leading treatment given to cancer patients. Chemotherapy involves one or more drugs that interfere with mitosis using non-specific intracellular toxins. Almost all of the side effects for this treatment adversely affect other rapidly-dividing cells in the body such as blood or stomach cells. Nearly all patients experience depression of the immune system while others may experience gastrointestinal distress, anemia, or vomiting.^[2] In addition, chemotherapy is not always effective due to cancer resistance or the blood brain barrier, prompting scientists to search for alternatives.^[3] Recently, naturally occurring peptides found in the venom of various animals have been observed to have selective cancer fighting properties, targeting only malignant cells while leaving healthy cells unharmed. If you need research peptides for your research work, make sure to only get it from a reliable supplier.
This study aims to create E. coli cells that have the ability to produce one such peptide; Polybia MP1, found in the Brazilian wasp, Polybia paulista, as a step to making MP1, a viable alternative to traditional chemotherapy. A gene fragment was developed by my team and myself to model the MP1 peptide. This gene fragment contains the MP1 gene sequence reverse translated and optimized for E. coli and the restriction sites EcoRI and NdeI for ligation. The MP1 gene fragment was ligated with the vector, which contained an ampicillin resistance gene to select for transformed colonies. Through observed colony growth and PCR, it was determined that the MP1 peptide can be successfully produced in E. coli cells, thus opening a new avenue of transgenic cancer therapies.

Introduction:

Targeted Cancer Therapies: With the advent of targeted cancer therapies over the past 30 years, the relative survival rate of patients has increased dramatically. However, many of these therapies only work for patients with very specific types of cancer and are therefore not a universal solution. One promising development is the possible use of venoms found naturally in animals to selectively target hallmark traits of cancer such as cell proliferation, angiogenesis, and metastasis while leaving healthy cells untouched. These types of treatments could tremendously improve the prognosis of cancer patients by working in conjunction with or even replacing current FDA-approved cancer treatments.^[4]
The venom of the Brazilian wasp (Polybia paulista) has been found to contain the protein Polybia MP1 (peptide sequence IDWKKLLDAAKQIL), also known as MP1.

Figure 1: Graph modified from Wang et al. showing how MP1 increases permeability in cancer cell membranes via the resulting leakage of cytoplasmic B-galactosidase. Cells treated with a higher concentration of polybia-MP1 cause more leakage, suggesting that MP1 has an ability to cause lysis.^[5]
MP1 has been shown to be a powerful cancer fighting toxin that selectively targets and destroys cancer cells while leaving healthy ones unharmed, according to Leite et al.^[6] It synergistically binds to two phospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), present in the outer leaflet of cancer cell membranes, perforating the cell and causing it to die by lysis.

Figure 2: The healthy cell (left) contains a normal phospholipid bilayer with transmembrane protein pumps as indicated by the red arrows. PS and PE lipids (represented by blue pentagons and green diamonds respectively) are pumped to the inner leaflet of the cell membrane. The cancer cell (right) does not have these protein pumps and therefore contains PS and PE phospholipids on the outer leaflet of the cell membrane.
Cancer cells lack these transmembrane proteins, and therefore have PS and PE molecules remain on the outer leaflet of their phospholipid bilayer. Leite et al. demonstrated that MP1 uses PS to bind to cancer cell membranes and PE to perforate the membranes, causing the cells to die.^[6] MP1 cannot bind to healthy cell membranes because they cannot access these lipids, which have been relocated to the inner leaflet of the cell instead of the outer via transmembrane protein pumps. Since it cannot bind to the membrane, MP1 cannot lyse healthy cells, and thus only targets cancer cells whilst leaving healthy cells unharmed.
MP1 in E. coli: E. coli does not naturally express the MP1 peptide but is a well understood, model organism that can be easily modified to produce a specific protein.^[7] Modifying E. coli to produce MP1 poses a challenge due to its short sequence of only 14 amino acids, which can be mistaken for food by E. coli cells.^[8] Two solutions to this problem were identified: tandem repeats where multiple copies of the gene sequence are coded into the gene fragment, with enterokinase sites between each repeat, and protein fusion with ubiquitin , where small peptides are ligated to a 8.6kDa inert, naturally occurring ubiquitin peptide.^[9,10] Research conducted at the Baltimore Underground Science Space (BUGSS) revealed that protein fusion with ubiquitin was the best solution to the problem. Tandem repeats have been shown to raise the pH of resulting peptides, which can inhibit the host organism’s ability to express the gene by changing the tertiary structure of enzymes involved in its transcription and translation.^[9] Ubiquitin is a natural peptide found in most organisms and serves to lengthen short proteins. In this study, ubiquitin is used as a fusion protein to lengthen the MP1 peptide and avoid being degraded by the host organism. (See Figure 3A).
pET-17b: As the MP1 gene is not native to the wild-type E. coli genome, it must be inserted into a suitable vector. Vectors are DNA sequences that work to transport foreign DNA into the recipient cell. For this study, the pET-17b vector was chosen to deliver the gene modification because it contains NdeI and EcoRI restriction sites as well as the beta-lactamase gene, which codes for a protein that confers resistance to the antibiotic ampicillin (See Figure 3B). These restriction sites were chosen due to availability and consistency of materials. Ampicillin resistance was necessary to selectively grow colonies of only transformed cells, which are cells that contain the pET-17b vector. A restriction digest of the MP1 gene and the pET-17b vector was performed as well as a subsequent ligation between the digested pieces of DNA. This ligated plasmid was then transformed into K 12 E. coli cells. The MP1 gene was reverse translated with the IDT tool, generating codons compatible with E. coli from the amino acid sequence of the original MP1 peptide. In 2016, a synthetic gene fragment was manufactured by Integrated DNA Solutions based on the design developed at BUGSS (See Figure 3A).
A.

B.

Figure 3: A. Diagram of MP1 gene fragment.^[11] The diagram shows (from left to right) EcoR1 restriction site, HIS-6, a region of histidine amino acids used to identify proteins during western blot assays, a ubiquitin fusion tag to extend the peptide, and the enterokinase gene coding for a protease to cleave the MP1 protein from the ubiquitin tag, the optimised MP1 gene, stop codon, and NdeI restriction site. B. Diagram of the pET-17b vector, including the ampicillin resistance gene, the NdeI restriction site, and the EcoRI restriction site (highlighted in yellow).

Methodology and Hypotheses:

The primary objective of this study is to demonstrate that the MP1 peptide can be produced by an E. coli cell. Based on the experimental design, it is expected that the transformation will produce colonies containing the recombinant plasmid that confers ampicillin resistance.
A synthetic gene fragment encoding the MP1 protein has been created from the MP1 peptide sequence IDWKKLLDAAKQIL. The gene fragment was modified with NdeI and EcoRI restriction sites for ligation with the pET-17b vector as well as a ubiquitin fusion tag. The gene fragment will be ligated with the pET-17b vector, and the resulting plasmid will be used to transform E. coli cells.
A.

B.

C. D.

Figure 4: Diagram detailing the transformation of E. coli cells. A) Full MP1 fragment showing EcoRI and NdeI restriction sites at each end of the sequence. B) MP1 insert digested by EcoRI and NdeI enzymes, resulting in sticky ends that facilitate ligation. C) Digested MP1 insert ligated with the digested pET-17b vector containing the ampicillin resistance gene. D) E. coli cells are made competent via salt shock prior to heat shock-mediated transformation, allowing the plasmids to enter the cell.
The resulting colonies will grow on ampicillin plates and be observed qualitatively. Once sufficient growth is achieved, the section of the plasmid containing the MP1 insert will be amplified through a polymerase chain reaction (PCR) process to verify that the MP1 peptide is being produced by the cell. The gene fragment will be isolated from samples confirmed to be expressing the MP1 gene and sequenced for further verification of successful transformation. A Western blot will be conducted in order to purify the MP1 peptide from E. coli cell homogenate.
Restriction sites were coded into each end of the MP1 gene fragment with some extra nucleotides to create an overhang so that the restriction enzymes had enough room to bind to their target sites and cleave the DNA. In this preparatory step, the restriction sites on the MP1 DNA gene fragment were cut with NdeI and EcoRI in order to create sticky ends. Two restriction enzymes are necessary in order to create a sticky end on each end of the insert fragment. Separate enzymes were used at each end to ensure that the insert would be ligated with the correct orientation. The pET-17b vector contained these restriction sites and was also digested by the same enzymes to create sticky ends complementary to those on the digested MP1 fragment.
Ligation: The digested vector and MP1 gene fragment were then ligated, resulting in the complete 3900 bp recombinant plasmid. Ligations were prepared in four solutions of differing insert:vector proportions (See Table 1) in order to determine which ligation would yield the highest rate of transformation.

Mixture number	Insert volume (μL)	Vector volume (μL)
1	2	2
2	2	3
3	3	2
4	3	3

Table 1: Volumes of vectors and insert fragments used in each ligation mixture. All ligations were brought up to a constant volume of 10 μL using distilled water. These solutions were then spread with aseptic techniques on ampicillin plates.
Transformation: K-12 E. coli were transformed with the ligated plasmid. To facilitate this, the E. coli samples were made competent (capable of accepting plasmids) through salt shock with calcium chloride solution(CaCl₂). The plasmid was placed in a solution containing competent E. coli cells and then heat shocked in a hot water bath at 42°C to allow the plasmid to pass through the cell membrane. The samples were spread on ampicillin plates so that only transformed cells would grow.
Positive and Negative Controls: In order to ensure the success of the transformation, a positive and a negative control were prepared. The negative control consisted of untransformed, wild-type K-12 E. coli cells. No growth was expected on this plate since E. coli cannot naturally survive in the presence of ampicillin. The positive control consisted of E. coli cells transformed with puc-19 control DNA. puc-19 is a control plasmid containing the gene for ampicillin resistance. Growth on these plates will confirm that the cells have been successfully transformed and are no longer ampicillin-sensitive.
Amplification and visualisation: To determine which samples contained the MP1 gene fragment previously ligated to the pET-17b vector before transformation, a PCR was conducted with primers specific to the 350 base pair fragment. 44 colonies were sampled from the resulting ampicillin plates to provide DNA for amplification. Gel electrophoresis was then conducted in order to visualize the results of the PCR. The PCR product of each sample was stained with purple loading dye and then loaded into 1% agarose gel stained with GelRed. Gels were imaged under UV light under which the stained DNA fluoresced. Samples with bands at approximately 350 base pairs were considered positive clones as the amplification region for the MP1 gene is approximately 350 base pairs in length.

Results:

The transformation showed growth on each of the four experimental plates as well as the positive control, but no growth on the negative control (See Figure 5). This indicates that transformation was successful; wild-type E. coli cannot grow in the presence of ampicillin as it is naturally ampicillin-sensitive. However, as mentioned previously, the pET-17b plasmid contains the beta-lactamase gene that degrades ampicillin; E. coli cells that grow successfully on the plates have expressed the plasmid and are resistant to ampicillin. Samples were allowed to grow past what is shown in Figure 4 until sufficient growth was achieved for PCR. Two days were allowed for clonal growth. Stocks were grown for each colony.
A B C

D E F

Figure 5: Solutions used on plates as listed in Table 1. Colonies on experimental plates are circled in red for clarity. Samples were allowed to grow beyond what is depicted in this figure in order to achieve sufficient growth for PCR.A. Plate containing sample of mixture 1. B. Plate containing sample of mixture 2. C. Plate containing sample of mixture 3. D. Plate containing sample of mixture 4. E. Negative control. F. Positive control.
Figure 5A shows that 3 colonies grew on the plate, showing that mixture 1 resulted in successful transformation. For all other mixtures, growth was also observed, although to a lesser extent. As expected, no growth was seen on the negative control (Figure 5E), confirming that wild-type E. coli is naturally sensitive to ampicillin. Positive control with puc-19 plasmid solution in Figure 5F shows considerable colony growth, which proves the validity of the transformation. While the 2:2 vector to insert ligation resulted in the most growth, all plates had a similar number of colonies so it is difficult to determine which mixture provided the highest transformation rate. Therefore, further repeats of the experiment are needed to generate mean colony counts and allow for more reliable conclusions.
PCR: 44 colonies were sampled from each transformation for PCR. The PCR product for each sample was analyzed using gel electrophoresis in order to determine which samples were observed to have amplicons, which are copies of the target 350bp gene fragment as in Figure 4. Seven samples were determined to have amplicons of this length.

Figure 6: Representative image of agarose gel used to qualitatively determine the success of the transformation. PCR product from each of the 44 sample colonies were run on an agarose gel. The first two lanes contain DNA ladders for size reference; all other lanes contained samples of the PCR product. The primers are indicated by the lowest band present in each lane. Lanes 9, 13,15, and 21 each presented bands approximately 350 base pairs in length (circled), suggesting the presence of the MP1 fragment. Several gels of this type were run to test each of the 44 PCR samples. A total of 7 samples were observed to produce bands at approximately 350 base pairs in length and were deemed successful.

Discussion:

Further research will include the creation of further experimental replicates to confirm the results of this study thus far. Each ligation yielded a similar number of colonies after transformation making it difficult to ascertain which in fact yields the highest transformation efficiency. Further replicates will allow for mean colony counts for each ligation to more accurately conclude which ligation is best. Additionally, due to time and resource restrictions, all 44 colonies were amplified in a single PCR instead of four individual PCRs with one for each ligation. Amplifying each ligation separately would allow for fluorescence analysis during gel visualization providing further insight into the effectiveness of each ligation.
Samples observed to yield PCR product at approximately 350 base pairs will be outsourced to a lab and sequenced. This step is to determine which of the samples are definitively expressing the recombinant MP1-containing plasmid. The final step is to conduct a Western blot in order to purify the MP1 protein itself. This entails running protein samples obtained from colonies previously determined via SDS-PAGE purification to be successfully expressing MP1. A HIS-6 epitope will be used to identify the peptide via binding of a specific antibody to show the successful expression of MP1 during Western Blot analysis.
The result of this study demonstrates that a potentially useful peptide naturally found in a species of wasp can be produced in E. coli cells, paving the way for potential in vivo approaches to cancer treatment. This suggests that chimeric organisms could be used to produce MP1 inside of the human body and deliver it specifically to tumor sites. MP1 has been shown to work particularly well with cancers such as leukemia^[6]. MP1 works similarly to many antimicrobial peptides, but stands out in the field of medicine due to its unique ability to specifically target cancer cells. This attribute has not been observed in any other antimicrobial peptides ^[4].
Utilizing the phospholipid bilayer in tackling cancer opens a wide range of possibilities for anticancer drugs previously untapped in scientific research. \”Cancer therapies that attack the lipid composition of the cell membrane would be an entirely new class of anticancer drugs,\” said Paul Beales from the University of Leeds. He continues, \”This could be useful in developing new combination therapies, where multiple drugs are used simultaneously by attacking different parts of the cancer cells at the same time.\” The production of MP1 in E. coli will improve the mechanism of delivery of this cancer-fighting protein. The ability to produce this peptide in vivo creates a host of possibilities for cancer treatments such as customized organisms with the ability to dissolve tumors.
As of 2018, cancer is the second leading cause of death in the world with 1 in every 6 deaths credited to the disease according to the World Health Organisation ^[1]. The modern technique of chemotherapy still has its limitations, taking a nonspecific approach to mitotic interference and causing adverse side effects. Chemotherapy also suffers from the problem of cancer resistance as cancer cells have rapidly adapted in order to survive and evade attack, by overproducing chemotherapy efflux pumps. These are transmembrane protein pumps that transport the treatment out of the cell^[3]. Targeted cancer therapies have been developed that specifically treat cancer cells, however, these are only effective for certain patients with specific genetic markers and will not work as a solution for all cancer patients. Nature has created more effective treatments in the form of venom peptides that have the ability to selectively target and destroy cancer cells^[3]. MP1 is one such venom and through biomimicry – the production of material modeled through biological processes – it can be utilized as a specific treatment for cancer that would have the benefits of current therapies without the pitfall of a limited scope of effectiveness. Peptide-based treatments have the potential to revolutionize the fight against cancer by serving as a targeted therapy available for the majority of the human population.

References:

[1] “Cancer.” World Health Organization. World Health Organization, February 5, 2019. https://www.who.int/cancer/en/.
[2] Rachel Airley. Cancer Chemotherapy, Chichester: Wiley-Blackwell, 2009.
[3] Emily Crowley, Christopher A. McDevitt, and Richard Callaghan. “Generating Inhibitors of P-Glycoprotein: Where to, Now?” Methods in Molecular Biology Multi-Drug Resistance in Cancer, May 2009, 405–32. https://doi.org/10.1007/978-1-60761-416-6_18.
[4] Mahadevappa, Ravikiran, Rui Ma, and Hang Fai Kwok. “Venom Peptides: Improving Specificity in Cancer Therapy.” Trends in Cancer 3, no. 9 (2017): 611–14. https://doi.org/10.1016/j.trecan.2017.07.004.
[5] Wang, Kairong, Jiexi Yan, Wen Dang, Xin Liu, Ru Chen, Jindao Zhang, Bangzhi Zhang, Wei Zhang, Ming Kai, Wenjin Yan, Zhibin Yang, Junqiu Xie, and Rui Wang. \”Membrane Active Antimicrobial Activity and Molecular Dynamics Study of a Novel Cationic Antimicrobial Peptide Polybia-MPI, from the Venom of Polybia Paulista.\” Peptides 39 (2013): 80-88. doi:10.1016/j.peptides.2012.11.002.
[6] Leite, Natália Bueno, Anders Aufderhorst-Roberts, Mario Sergio Palma, Simon D. Connell, João Ruggiero Neto, and Paul A. Beales. “PE and PS Lipids Synergistically Enhance Membrane Poration by a Peptide with Anticancer Properties.” Biophysical Journal 109, no. 5 (2015): 936–47. https://doi.org/10.1016/j.bpj.2015.07.033.
[7] Li, Yifeng. “Erratum to ‘Recombinant Production of Antimicrobial Peptides in Escherichia Coli: A Review’ [Protein Express. Purif. 80 (2011) 206–267].” Protein Expression and Purification 82, no. 1 (2012): 252. https://doi.org/10.1016/j.pep.2011.11.006.
[8] Li, Yan, Jiarong Wang, Jing Yang, Chanjuan Wan, Xiaoming Wang, and Hongbin Sun. “Recombinant Expression, Purification and Characterization of Antimicrobial Peptide ORBK in Escherichia Coli.” Protein Expression and Purification 95 (2014): 182–87. https://doi.org/10.1016/j.pep.2013.12.011.
[9] Kuliopulos, Athan, and Christopher T. Walsh. “Production, Purification, and Cleavage of Tandem Repeats of Recombinant Peptides.” Journal of the American Chemical Society116, no. 11 (1994): 4599–4607. https://doi.org/10.1021/ja00090a008.
[10] Baker, Rohan T. “Protein Expression Using Ubiquitin Fusion and Cleavage.” Current Opinion in Biotechnology7, no. 5 (1996): 541–46. https://doi.org/10.1016/s0958-1669(96)80059-0.
[11] Image taken from: http://www.youbio.cn/product/vt1194

Acknowledgements

Special thanks to Sarah Laun PhD and the team at the Baltimore Underground Science Space Tom Burkett PhD, Lisa Scheifele PhD, and Ryan Hammond.

About the Author

Alex Misiaszek is a Junior at St. Albans School in Washington, DC.  He is passionately interested in Molecular Biology and has been pursuing independent research at the Baltimore Underground Science Space since he was in Seventh Grade. He is eager to share what he has learned from his research into the use of E. coli cells that have been genetically modified to produce a cancer fighting toxin.