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E.Cotector: The Fluorescent E. coli with Surface Displayed Anti-Cancer Marker scFv to Detect Specific Cancer Markers
Fung-Ling Ng¶ (1), Kuan-Chun Lan¶ (1), Chen-Yu Chang (1), Chih-Hsuan Hsu (1), Tsung-Yu Ho (1), Ming-Hsiu Hsieh (1), Ting-Han Kuo (1), Yu-Yun Wang (2), Ru-Huah Lin (1), Wei-Hung Hsu (3), Yu-Ting Lin (4), Ya-Chih Tai (1), Rui-Xing Wang (3), Nai-Chen Chi (3), Yu-An Chen (1), Yang-Chen Lin (1), Tzu-Yin Wei (1), Jing-Chin Lin (1), Hsiao-Ching Lee (1), Wen-Liang Chen* (1)
1 Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan
2 Department of Applied Chemistry, National Chiao Tung University, Hsinchu, Taiwan
3 Undergraduate Honors Program of Nano Science Technology, National Chiao Tung University, Hsinchu, Taiwan
4 Department of Transportation and Logistics Management, National Chiao Tung University, Hsinchu, Taiwan
*Corresponding author: Wen-Liang Chen (firstname.lastname@example.org)
¶These authors contributed equally to this work
Conceptualization: Fung-Ling Ng, Yu-An Chen, Chen-Yu Chang, Chih-Hsuan Hsu, Ming-Hsiu Hsieh, Jing-Chin Lin, Ru-Huah Lin, Ting-Han Kuo, Rui-Xing Wang, Tzu-Yin Wei, Yu-Yun Wang, Nai-Chen Chi, Wei-Hung Hsu, Yang-Chen Lin, Ya-Chih Tai, Yu-Ting Lin, Wen-Liang Chen
Methodology: Wen-Liang Chen, Ru-Huah Lin, Yu-Yun Wang
Investigation: Chen-Yu Chang, Chih-Hsuan Hsu, Tsung-Yu Ho, Ming-Hsiu Hsieh, Ru-Huah Lin, Ting-Han Kuo, Yu-Yun Wang, Ya-Chih Tai
Writing: Fung-Ling Ng, Kuan-Chun Lan
Funding Acquisition: Wen-Liang Chen
Supervision: Wen-Liang Chen, Hsiao-Ching Lee
Monitoring of cancer markers at certain intervals throughout the treatments provides important information for targeted drugs prescribing procedure. It can ensure the accuracy and efficacy of targeted drugs dosages and achieve the goal of individualize treatment. Furthermore, in a more advanced monitoring process, a multiple markers detection platform could provide reference for combination therapy. In this study, we developed a cancer marker monitoring platform that constructed by a dual-expression system in Escherichia coli. This system consisted of anti-cancer marker in single chain variable fragment (scFv) form and fluorescent protein as a cancer markers monitoring platform. The anti-cancer markers scFv on E. coli surface are originated from the FDA approved monoclonal antibody targeted drugs which are the anti-HER2, anti-EGFR and anti-VEGF. We constructed these E. coli with different fluorescence, as we called them the E.Cotector for future utilizing in multiple markers detection method. In addition, the fluorescent expression of E. coli enables the monitoring observation to be more convenient. Furthermore, to utilize the platform in biosensor, we verified the function of gold binding polypeptide (GBP) as a biological adhesive that enable E.Cotector to immobilize on the gold chip of biosensor and could be applied in detection field in the future.
The laboratory expenses were supported by the team instructor, Professor Wen-Liang Chen. Other funding was from the National Chiao Tung University, the Department of Biological Science and Technology.
The authors declare that no competing interests exist.
All data are fully available without restriction.
According to the prediction of the World Health Organization GloboCan, the global cancer cases will increase tremendously from 15 million cases in 2015 to 24 million cases in 2035. Compared with the traditional chemotherapy, targeted therapies would be more accurate to targeted markers on cancer cells. Furthermore, in between 1990s until 2013, the FDA’s new drug approvals for targeted therapies have increased from 5% to 45%, which indicates the high developmental of targeted therapies in recent cancer treatment. The development of targeted therapies also makes it increasingly possible to provide individualized and personalized medical therapy by identifying the patients. These therapies such as hormone therapy, immunotherapy and gene therapy may regulate the signal transduction, inhibit cell growth and enhance apoptosis of cancer cells.
To achieve the goal of personalized treatment, the monitoring of cancer markers in advanced companion diagnostic test throughout the whole treatment has become crucial. Through the diagnostic test, doctors can obtain detailed information during determination of types and dosages of specific targeted drugs. Until the May of 2014, the FDA has approved 26 packages of diagnostic devices corresponding to the specific targeted drugs and those respectively cancer markers will be monitored. For example, the detection of overexpressed HER2 biomarkers before the usage of Herceptin targeted drug for metastatic breast cancer patients; Erbitux targeted drugs for overexpressed EGFR such as in colorectal cancer, etc. [4-6]
In this study, we developed a multiple cancer markers diagnostic method for targeted drug therapies. We designed a dual protein expression system that contains anti-cancer markers and multiple fluorescent signals in E. coli that we called them as the E.Cotector. The targeted markers are the overexpressed VEGF, EGFR and HER2 that are closely related to tumor growth. The respectively anti-cancer markers are the FDA approved monoclonal antibody targeted drugs: Bevacizumab (Avastin® anti-VEGF), Cetuximab (Erbitux® anti-EGFR) and Trastuzumab (Herceptin® anti-HER2). By using the expression system, we would like to provide drug prescribing information after detecting multiple markers directly with E.Cotector. Moreover, reference for prescribing of combination therapy can be obtained by utilizing different kinds of fluorescent E.Cotector for multiple markers detection. Furthermore, we also developed to use this system as a probe in biosensor. We co-expressed the GBP (gold binding polypeptide) on the surface of E. coli. The idea of combining the designed E. coli as biological recognition part and the gold chip to act as the signal transducer part could be applied in the diagnostic and drug monitoring stage before treatment in a more precise way.
Materials and methods
Bacterial strain, vector and cultural condition
E. coli BL21 was used as strain for the construction of dual protein expression system. The vector pSB1C3 (iGEM) was used for all the genes expression. The strain was grown in LB medium (120rpm, 12-16 hours) and 25 μg/ml of chloramphenicol was added to the media for selection as needed.
Selection and redesign of anti-cancer markers into scFv
Anti-cancer marker drugs selected for this study are FDA approved. They were the anti-VEGF (BBa_K1694023) which targeted the vascular endothelial growth factor (VEGF); the anti-EGFR (BBa_K1694024) which targeted the epidermal growth factor receptor (EGFR); the anti-HER2 (BBa_K1694025) which targeted the Human epidermal growth factor receptor (HER2). We further redesigned the sequences of these antibodies drugs into single chain variable fragment (scFv) by linking both the heavy chain and light chain of variable region of anti-cancer marker with a peptide linker. In order to be possibly expressed by E. coli, we redesigned the sequences of these antibodies drugs into single chain variable fragment (scFv) form, as it is only 20 percent of the size of most normal antibodies while maintaining the function of binding with specific antigens.
Anchoring the scFv on the outer membrane of E. coli
The transmembrane protein selected for this study was Lpp-OmpA (BBa_K1694002) which consisted of the signal sequence of Lpp, the first nine N-terminal amino acids of Lpp and the residues 46-159 of OmpA. The Lpp of this fusion transmembrane protein targets the protein on the membrane while the transmembrane domain of OmpA serves as an anchor. By the external exposed loops of the C-terminal of OmpA, scFv which designed at behind of Lpp-OmpA can be easily anchored to the outer membrane. Between the OmpA and scFv, we also designed a cut site of restriction enzyme called NcoI allowing the linked scFv to be replaced conveniently.
Co-expression of fluorescent protein
To improve and optimize the function of E. coli as a cancer-markers detector, we co-transformed the genes of fluorescent proteins to act as a reporter after detection. The fluorescent genes used in this study were red fluorescent (rfp) (BBa_E1010) and green fluorescent (gfp) (BBa_E0040).
Gene construction of scFv and cell staining with E.Cotectors
We performed electrophoresis to verify the construction of scFv genes into the E. coli BL21. As we used VF2 and VR primers which flank the BioBrick cloning site and need to add approximately 200 nucleotides to the PCR product length, each DNA length will increase around 200bp.
Cancer cell lines used to test the targeting and binding ability of the E.Cotector was SKOV-3 from Bioresource Collection and Research Center (BCRC). SKOV-3 is a human ovarian cancer cell line, which overexpresses EGFR, VEGF and HER2. We cultivated the staining E. coli for 16~18 hours. Before staining the cells, we used 4% paraformaldehyde to fix the cells on dish. After staining the cancer cells in dishes with different types of E. coli, we washed out the unbinding E. coli with Phosphate-buffered saline (PBS) solution. We observed the binding ability of E.Cotector under fluorescent microscope (400X).
Further application— Gold Binding Polypeptide (GBP)
Gene construction of GBP
Gold binding polypeptide (GBP) (BBa_K1694027) was designed with three-repeated specific 14 amino acids sequences: [MHGKTQATSGTIQS]. By using Molecular Dynamics (MD), it indicated that GBP, with an antiparallel β-sheet structure, could recognize gold surface via OH-binding. It is likely that the ligands on GBP recognized the atomic lattice of gold, aligning the molecule along the variants of a six-fold axis on the Au (III) surface. To transfer GBP to the outer membrane of E. coli, there was a gene design of FadL transmembrane protein linked before the GBP gene.
The FadL protein is an outer membrane protein of the E. coli. With its twenty antiparallel β-strands structure, the β-barrel of FadL protein is formed and is connected by 9 internal and 10 external loops. Due to the β-structure, the FadL protein is able to cross the outer membrane multiple times to form a long-chain fatty acid-specific channel. In this study, we encoded the first 384 amino acids of FadL from N-terminus to display of GBP on the E. coli cell surface.
We performed electrophoresis to verify the construction of FadL-GBP gene into the E. coli BL21. As we used VF2 and VR primers which flank the BioBrick cloning site and need to add approximately 200 nucleotides to the PCR product length, the DNA length will increase around 200bp.
Validation of GBP function
To verify the function of GBP, we designed one type of E. coli that simultaneously produces both GBP and gfp as experimental groups, and the other type of E. coli only with green fluorescent as control group. Besides, the gold chips used in this study are those used in biosensor as signal transducer with size of 6mm*3mm (Fig 5A). We put gold chip into each eppendorf that contained the prepared E. coli solution and kept the eppendorfs in the incubator at 25 degree Celfsius. After that, we took out the gold chips and immersed them into new eppendorfs with Phosphate-buffered saline (PBS) solution. To wash out the unbinding E. coli completely, we used rotary machine to rotate the eppendorfs in 360。.Then, we placed those washed gold chips on the slides to observe the binding efficiency of E. coli by the fluorescent microscope (Fig 5B).
Gene constructions of anti-cancer markers scFv
The three anti-cancer markers that have been completely constructed were the anti-VEGF, anti-EGFR and anti-Her2. Each of the BioBrick construction includes the conservative promoter, RBS, Lpp-ompA and anti-cancer marker. The DNA length of anti-VEGF is 1256bp; anti-EGFR is 1244bp; anti-HER2 is 1089bp. After amplification with PCR, the PCE products have length of 1300bp~1500bp, which anti-VEGF (Fig A1), anti-EGFR (Fig B1) and anti-HER2 (Fig C1).
Binding ability of E.Cotectors to cancer markers on cancer cell line
We used (SKOV-3) cancer cells in this study as this type of cells overexpresses all the three cancer-markers, which are the EGFR, VEGF and Her2. By staining the cells with the three types E.Cotector, we could validate the binding ability of each E.Cotector. We used fluorescent microscope (400 X magnifications) to observe the result of cell staining. In the study, we created two different kinds of fluorescent E.Cotector, they are red and green fluorescent as shown in Fig 7. As result shown, E.Cotector have successfully targeted the cancer markers on cancer cells respectively and being observed which shown the co-expression is worked.
Expression of gold-binding polypeptide on outer membrane E. coli
The inserted DNA of the completed FadL-GBP is around 1345bp. After amplification with PCR, the PCR product have length of 1500~1700bp (Fig A1). As result, the constructions of BioBrick were correct.
To investigate the function and effectiveness of GBP on gold binding ability of E. coli, we immersed gold chips in E. coli solution. To observe the binding ability of E. coli clearly under fluorescent microscope (400X magnification), we transformed GBP in green fluorescent E. coli. In the result, there were green fluorescent E. coli (with GBP) binding on the gold chips (Fig B2) compared with the control group there is no E. coli binding on the gold chips (Fig B1). By this observation, we concluded primarily that GBP, which are expressed on the outer membrane of E. coli, were possible to aid E. coli to bind on gold chip.
Companion diagnosis is important throughout the targeted drug treatments. Thus, scientists have tried hard to develop targeted drug monitoring methods that could detect several markers simultaneously with higher sensitivity and efficiency. In our study, we chose three widely studied cancer markers EGFR, HER2 and VEGF as the validated targets for cancer therapy. We redesigned three kinds of FDA-approved monoclonal antibody targeted drugs: Cetuximab (Erbitux® anti-EGFR), Trastuzumab (Herceptin® anti-HER2) and Bevacizumab (Avastin® anti-VEGF) into scFv form to target those markers. Current studies demonstrated to functionally display scFv on the cell or E. coli surface with different kinds of membrane protein or transmembrane domain such as PDGFR or B7-1 transmembrane domain (Irina V. Balyasnikova et al.,2010), autotransporter β-barrel domain (Lin-XuWang et al., 2014), and the Lpp-OmpA (George Georgiou et al., 1996; Patrick S. Daugherty et al., 1999). [9-12] In our design, we modified the surface display system of anti-cancer scFv, which composed of lipoprotein (Lpp) and outer membrane protein A (OmpA). (Fig 3) Since the function of Lpp is to send the targeted protein to the outer membrane and OmpA serves as an anchor on the membrane, Lpp-OmpA has great advantage in high surface display efficiency. [12-15]
Previous study indicated that the scFv fused with fluorescent proteins could be successfully produced in E. coli and the function of purified scFv was proved by immunostaining.  We also demonstrated the fluorescent E.Cotector functionally binding EGFR, VEGF and Her2 on SKOV-3 human ovarian cancer cell. (Fig 7) In our system, multiple fluorescent E.Cotector could be used to target multiple markers simultaneously. Therefore, a direct reference for health care professional on prescribing a personalized combination therapy.
In previous researches, the application of GBP as a connection between the transducer part (gold chip) and bio-receptor part in biosensor has been proved to be a practical method. In addition, method that directly using whole cell dual expression system link with transducer part was demonstrated to be useful for developing whole-cell biosensor chips. In our design, the transmembrane protein, FadL, fused with the GBP, is capable of being an anchoring motif to display GBP on the surface of E. coli and cause E. coli bind on the gold chip. In our study, the green fluorescent E. coli with GBP expressed on outer membrane showed the ability of E. coli to bind on gold chip. (Fig 8B) For high analyte binding of biosensor, the oriented immobilization of detective elements on transducer surface to display their antigen-binding domains is a crucial issue. Previous researches demonstrates the way utilizing GBP to immobilize detective element on the surface of biosensor, we also believe that our E. coli with surface display scFv could potentially achieve oriented immobilization with GBP expression.[7, 19]
In conclusion, our study had successfully using engineered E. coli to co-express both the anti-cancer marker and fluorescent protein simultaneously for diagnostic application. In addition, the further utilizing GBP co-expressed E. coli showed the ability of binding on the gold surface. We hope that this system could pave the way for building targeted drug monitor platform and being applied in biosensor in the future.
The authors would like to thank the team instructors, Wen-Liang Chen and Hsiao-Ching Lee for their fully support throughout the whole project included the guidance of experimental designs and team operation for this year. Moreover, we also appreciate the helping from the previous team members of NCTU_Formosa, Lung-Chieh Chen, Chun-San Tai, Malvin Jefri, Goang-Jiun Wang, Chun-Wei Chiu for giving us essential advises that made our project more fluent. We thank Bobo Huang for helping the team in the process of cell staining. We thank Ming-Ju Tsai for helping us in performing the related model of our project. Furthermore, we would like to thank Huai-En Lu and Rachel Lee for presenting our project in a more professional way. Lastly, we would like to thank all the people who supported the team in 2015.
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