Feb 12, 2024
Version 1
Recombinant retroviral vectors that express EGF, TGF alpha, NRG2 beta, and the NRG2 beta Q43L mutant V.1
- 1Auburn University;
- 2University of Alabama-Birmingham
Protocol Citation: Ella Wilson, Markelle Scott, Vipasha Dwivedi, David J Riese II, Madison Zelan 2024. Recombinant retroviral vectors that express EGF, TGF alpha, NRG2 beta, and the NRG2 beta Q43L mutant. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx3d6og8j/v1Version created by David J Riese II
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 10, 2024
Last Modified: February 12, 2024
Protocol Integer ID: 94983
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Abstract
Here we describe the construction of recombinant retroviral expression vectors based on pLXSN-HygR that drive the ectopic expression of the Epidermal Growth Factor (EGF), Transforming Growth Factor alpha (TGFalpha), Neuregulin 2beta (NRG2beta), or the NRG2beta Q43L mutant protein.
Introduction
Introduction
Elevated signaling by members of the epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases contributes to numerous human malignancies. This elevated signaling may be due to gain-of-function mutations in the receptor genes, increased receptor gene transcription, or elevated ligand expression [1-11]. Because these receptors are tractable targets for therapeutic intervention [12-32], there is much interest in tools that can be used to study these mechanisms of elevated signaling and the biological consequences of elevated signaling.
Here we describe the construction of recombinant retroviral expression vectors based on pLXSN-HygR (
pLXSN-HygR.dna56KB ) [33] that drive the ectopic expression of the Epidermal Growth Factor (EGF) [34], Transforming Growth Factor alpha (TGFalpha) [34], Neuregulin 2beta (NRG2beta) [35-40], or the NRG2beta Q43L mutant protein [39, 40].
pLXSN-HygR.dna56KB ) [33] that drive the ectopic expression of the Epidermal Growth Factor (EGF) [34], Transforming Growth Factor alpha (TGFalpha) [34], Neuregulin 2beta (NRG2beta) [35-40], or the NRG2beta Q43L mutant protein [39, 40].Methods
Methods
Construction of pLXSN-HygR-EGF
We have previously described the construction of pENTR-EGF-Short (pENTR-EGF-Short.dna32KB ) [34]. This plasmid encodes the soluble, mature form of EGF. This EGF coding sequence is flanked in frame on the 5’ end by a sequence that encodes a BiP signal sequence to facilitate protein trafficking. The EGF coding sequence is flanked in frame on the 3’ end by V5 and His6 tags to facilitate immunodetection and purification. The sequence of the entire BiP-EGF-V5-His6 fusion protein is shown in Figure 1 (below).
pENTR-EGF-Short.dna32KB ) [34]. This plasmid encodes the soluble, mature form of EGF. This EGF coding sequence is flanked in frame on the 5’ end by a sequence that encodes a BiP signal sequence to facilitate protein trafficking. The EGF coding sequence is flanked in frame on the 3’ end by V5 and His6 tags to facilitate immunodetection and purification. The sequence of the entire BiP-EGF-V5-His6 fusion protein is shown in Figure 1 (below).The BiP sequence is underlined, the EGF sequence is doubly underlined, and the V5 sequence is underlined.
We amplified the BiP-EGF-V5-His6 coding sequence from pENTR-EGF-Short using primers Modified-BiP-Primer and Modified-His6-Primer, as shown in Figure 2 (below – also see pENTR-EGF-Short.dna32KB ).
pENTR-EGF-Short.dna32KB ). The resulting amplicon is predicted to be approximately 328 bp in length and contain a unique XhoI site immediately upstream of the BiP coding sequence and a unique BamHI site immediately downstream of the His6 coding sequence. Thus, the amplicon was digested with XhoI and BamHI and was ligated to the 6412 bp fragment of pLXSN-HygR-ERBB2 pLXSN-HygR-ERBB2.dna72KB ), which encodes the pLXSN-HygR vector sequences. We used standard molecular biology techniques to complete this subcloning. Candidate clones were validated by restriction mapping and next-generation DNA sequencing (NGS), resulting in pLXSN-HygR-EGF (pLXSN-HygR-EGF.dna53KB ).
pLXSN-HygR-ERBB2.dna72KB ), which encodes the pLXSN-HygR vector sequences. We used standard molecular biology techniques to complete this subcloning. Candidate clones were validated by restriction mapping and next-generation DNA sequencing (NGS), resulting in pLXSN-HygR-EGF (pLXSN-HygR-EGF.dna53KB ).Construction of pLXSN-HygR-TGFalpha-Short
We have previously described the construction of pENTR-TGFalpha-Short (pENTR-TGFalpha-Short.dna30KB ) [34]. This plasmid encodes the soluble, mature form of TGFalpha. This TGFalpha coding sequence is flanked in frame on the 5’ end by a sequence that encodes a BiP signal sequence to facilitate protein trafficking. The TGFalpha coding sequence is flanked in frame on the 3’ end by V5 and His6 tags to facilitate immunodetection and purification. The sequence of the entire BiP-TGFalpha-Short-V5-His6 fusion protein is shown in Figure 1 (above). The BiP sequence is underlined, the TGFalpha-Short sequence is doubly underlined, and the V5 sequence is underlined.
pENTR-TGFalpha-Short.dna30KB ) [34]. This plasmid encodes the soluble, mature form of TGFalpha. This TGFalpha coding sequence is flanked in frame on the 5’ end by a sequence that encodes a BiP signal sequence to facilitate protein trafficking. The TGFalpha coding sequence is flanked in frame on the 3’ end by V5 and His6 tags to facilitate immunodetection and purification. The sequence of the entire BiP-TGFalpha-Short-V5-His6 fusion protein is shown in Figure 1 (above). The BiP sequence is underlined, the TGFalpha-Short sequence is doubly underlined, and the V5 sequence is underlined.We amplified the BiP-TGFalpha-Short-V5-His6 coding sequence from pENTR-TGFalpha-Short using primers Modified-BiP-Primer and Modified-His6-Primer (Figure 2 above– also see pENTR-TGFalpha-Short.dna30KB ). The resulting amplicon is predicted to be approximately 319 bp in length and contain a unique XhoI site immediately upstream of the BiP coding sequence and a unique BamHI site immediately downstream of the His6 coding sequence. Thus, the amplicon was digested with XhoI and BamHI and was ligated to the 6412 bp fragment of pLXSN-HygR-ERBB2 (pLXSN-HygR-ERBB2.dna72KB ) which encodes the pLXSN-HygR vector sequences. We used standard molecular biology techniques to complete this subcloning. Candidate clones were validated by restriction mapping and next-generation DNA sequencing (NGS), resulting in pLXSN-HygR-TGFalpha-Short (pLXSN-HygR-TGFalpha-Short.dna53KB ).
pENTR-TGFalpha-Short.dna30KB ). The resulting amplicon is predicted to be approximately 319 bp in length and contain a unique XhoI site immediately upstream of the BiP coding sequence and a unique BamHI site immediately downstream of the His6 coding sequence. Thus, the amplicon was digested with XhoI and BamHI and was ligated to the 6412 bp fragment of pLXSN-HygR-ERBB2 (pLXSN-HygR-ERBB2.dna72KB ) which encodes the pLXSN-HygR vector sequences. We used standard molecular biology techniques to complete this subcloning. Candidate clones were validated by restriction mapping and next-generation DNA sequencing (NGS), resulting in pLXSN-HygR-TGFalpha-Short (pLXSN-HygR-TGFalpha-Short.dna53KB ).Construction of pLXSN-HygR-NRG2beta and pLXSN-HygR-NRG2beta-Q43L
We have previously described the construction of pMT-BiP-NRG2beta-V5-His6 (pMT-BiP-NRG2beta-V5-His6.dna46KB ) [36, 37]. This plasmid encodes the soluble, mature form of NRG2beta. This NRG2beta coding sequence is flanked in frame on the 5’ end by a sequence that encodes a BiP signal sequence to facilitate protein trafficking. The NRG2beta coding sequence is flanked in frame on the 3’ end by V5 and His6 tags to facilitate immunodetection and purification. The sequence of the entire BiP-NRG2beta-V5-His6 fusion protein is shown in Figure 1 (above). The BiP sequence is underlined, the NRG2beta sequence is doubly underlined, and the V5 sequence is underlined.
pMT-BiP-NRG2beta-V5-His6.dna46KB ) [36, 37]. This plasmid encodes the soluble, mature form of NRG2beta. This NRG2beta coding sequence is flanked in frame on the 5’ end by a sequence that encodes a BiP signal sequence to facilitate protein trafficking. The NRG2beta coding sequence is flanked in frame on the 3’ end by V5 and His6 tags to facilitate immunodetection and purification. The sequence of the entire BiP-NRG2beta-V5-His6 fusion protein is shown in Figure 1 (above). The BiP sequence is underlined, the NRG2beta sequence is doubly underlined, and the V5 sequence is underlined.We amplified the BiP-NRG2beta-V5-His6 coding sequence from pMT-BiP-NRG2beta-V5-His6 using primers Modified-BiP-Primer and Modified-His6-Primer (Figure 2 above – also see pMT-BiP-NRG2beta-V5-His6.dna46KB ). The resulting amplicon is predicted to be approximately 373 bp in length and contain a unique XhoI site immediately upstream of the BiP coding sequence and a unique BamHI site immediately downstream of the His6 coding sequence. Thus, the amplicon was digested with XhoI and BamHI and was ligated to the 6412 bp fragment of pLXSN-HygR-ERBB2 (pLXSN-HygR-ERBB2.dna72KB ), which encodes the pLXSN-HygR vector sequences. We used standard molecular biology techniques to complete this subcloning. Candidate clones were validated by restriction mapping and next-generation DNA sequencing (NGS), resulting in pLXSN-HygR-NRG2beta (pLXSN-HygR-NRG2beta.dna54KB ).
pMT-BiP-NRG2beta-V5-His6.dna46KB ). The resulting amplicon is predicted to be approximately 373 bp in length and contain a unique XhoI site immediately upstream of the BiP coding sequence and a unique BamHI site immediately downstream of the His6 coding sequence. Thus, the amplicon was digested with XhoI and BamHI and was ligated to the 6412 bp fragment of pLXSN-HygR-ERBB2 (pLXSN-HygR-ERBB2.dna72KB ), which encodes the pLXSN-HygR vector sequences. We used standard molecular biology techniques to complete this subcloning. Candidate clones were validated by restriction mapping and next-generation DNA sequencing (NGS), resulting in pLXSN-HygR-NRG2beta (pLXSN-HygR-NRG2beta.dna54KB ).We constructed pLXSN-HygR-NRG2beta-Q43L, which encodes the Q43L mutant of NRG2beta, in an identical manner, except we used pMT-BiP-NRG2beta-Q43L-V5-His6 (pMT-BiP-NRG2beta-Q43L-V5-His6.dna46KB ) as the template for the PCR amplification reaction. The sequence of the BiP-NRG2beta-Q43L-V5-His6 fusion protein is provided in Figure 2 (above). The Q43L mutant is indicated by red text and yellow highlighting. The sequence of pLXSN-HygR-NRG2beta is provided in pLXSN-HygR-NRG2beta-Q43L.dna53KB .
pMT-BiP-NRG2beta-Q43L-V5-His6.dna46KB ) as the template for the PCR amplification reaction. The sequence of the BiP-NRG2beta-Q43L-V5-His6 fusion protein is provided in Figure 2 (above). The Q43L mutant is indicated by red text and yellow highlighting. The sequence of pLXSN-HygR-NRG2beta is provided in pLXSN-HygR-NRG2beta-Q43L.dna53KB .References
References
1. Androutsopoulos, G., et al., The ErbB Signaling Network and Its Potential Role in Endometrial Cancer. Epigenomes, 2023. 7(4).
2. Palumbo, C., et al., Recent findings on the impact of ErbB receptors status on prognosis and therapy of head and neck squamous cell carcinoma. Front Med (Lausanne), 2023. 10: p. 1066021.
3. Vulf, M., et al., NGR4 and ERBB4 as Promising Diagnostic and Therapeutic Targets for Metabolic Disorders. Front Biosci (Elite Ed), 2023. 15(2): p. 14.
4. Rodriguez, S.M.B., et al., An Overview of EGFR Mechanisms and Their Implications in Targeted Therapies for Glioblastoma. Int J Mol Sci, 2023. 24(13).
5. Liu, M., et al., Germline EGFR mutations in lung cancer (Review). Oncol Lett, 2023. 26(1): p. 282.
6. Sentana-Lledo, D., et al., EGFR exon 20 insertion mutations and ERBB2 mutations in lung cancer: a narrative review on approved targeted therapies from oral kinase inhibitors to antibody-drug conjugates. Transl Lung Cancer Res, 2023. 12(7): p. 1590-1610.
7. Galve-Calvo, E., et al., Narrative Review of Multidisciplinary Management of Central Nervous Involvement in Patients with HER2-Positive Metastatic Breast Cancer: Focus on Elderly Patients. Adv Ther, 2023. 40(8): p. 3304-3331.
8. McNamara, B., et al., HER2 Oncogene as Molecular Target in Uterine Serous Carcinoma and Uterine Carcinosarcoma. Cancers (Basel), 2023. 15(16).
9. Ayasun, R., M. Ozer, and I. Sahin, The Role of HER2 Status in the Biliary Tract Cancers. Cancers (Basel), 2023. 15(9).
10. Loeffler, E., et al., HER2 Alterations in Non-Small Cell Lung Cancer: Biologico-Clinical Consequences and Interest in Therapeutic Strategies. Life (Basel), 2023. 14(1).
11. Yang, L.L., et al., The treatment of patients with non-small cell lung cancer carrying uncommon EGFR mutations, HER2 mutations, or brain metastases: a systematic review of pre-clinical and clinical findings for dacomitinib. Transl Cancer Res, 2023. 12(8): p. 2197-2211.
12. Shaban, N., et al., Targeted Inhibitors of EGFR: Structure, Biology, Biomarkers, and Clinical Applications. Cells, 2023. 13(1).
13. Li, P., et al., The next generation of EGFR inhibitors: a patenting perspective of PROTACs based EGFR degraders. Expert Opin Ther Pat, 2023. 33(7-8): p. 477-492.
14. Halder, S., et al., Targeting the EGFR signaling pathway in cancer therapy: What’s new in 2023? Expert Opin Ther Targets, 2023. 27(4-5): p. 305-324.
15. Chen, Q., et al., Targeting HER3 to overcome EGFR TKI resistance in NSCLC. Front Immunol, 2023. 14: p. 1332057.
16. Zubair, T. and D. Bandyopadhyay, Small Molecule EGFR Inhibitors as Anti-Cancer Agents: Discovery, Mechanisms of Action, and Opportunities. Int J Mol Sci, 2023. 24(3).
17. Dhiwar, P.S., et al., An assessment of EGFR and HER2 inhibitors with structure activity relationship of fused pyrimidine derivatives for breast cancer: a brief review. J Biomol Struct Dyn, 2023: p. 1-18.
18. Crintea, A., et al., Targeted EGFR Nanotherapy in Non-Small Cell Lung Cancer. J Funct Biomater, 2023. 14(9).
19. Pan, Q., et al., Recent Advances in Boosting EGFR Tyrosine Kinase Inhibitors-Based Cancer Therapy. Mol Pharm, 2023. 20(2): p. 829-852.
20. Kasi, P.M., et al., Anti-EGFR Antibodies in the Management of Advanced Colorectal Cancer. Oncologist, 2023. 28(12): p. 1034-1048.
21. Qu, M., et al., Advances in HER2-Targeted Treatment for Advanced/Metastatic Urothelial Carcinoma. Bladder (San Franc), 2023. 10: p. e21200012.
22. Taurelli Salimbeni, B., et al., Innovative Therapeutic Approaches for Patients with HER2-Positive Breast Cancer. Cancer Treat Res, 2023. 188: p. 237-281.
23. Fernandes, C.L., D.J. Silva, and A. Mesquita, Novel HER-2 Targeted Therapies in Breast Cancer. Cancers (Basel), 2023. 16(1).
24. Angelis, V. and A.F.C. Okines, Systemic Therapies for HER2-Positive Advanced Breast Cancer. Cancers (Basel), 2023. 16(1).
25. Zheng-Lin, B., R.P. Graham, and T.S. Bekaii-Saab, Targeting ERBB2/HER2 genetic alterations: an expanding therapeutic opportunity in gastrointestinal cancers. Chin Clin Oncol, 2023. 12(5): p. 55.
26. Graff, S.L., F. Yan, and Y. Abdou, Newly Approved and Emerging Agents in HER2-Positive Metastatic Breast Cancer. Clin Breast Cancer, 2023. 23(7): p. e380-e393.
27. Stanowicka-Grada, M. and E. Senkus, Anti-HER2 Drugs for the Treatment of Advanced HER2 Positive Breast Cancer. Curr Treat Options Oncol, 2023. 24(11): p. 1633-1650.
28. Nakano, K., The Future of HER2-Targeted Treatment for Osteosarcoma: Lessons from the Negative Trastuzumab Deruxtecan Results. Int J Mol Sci, 2023. 24(23).
29. Pous, A., et al., HER2-Positive Gastric Cancer: The Role of Immunotherapy and Novel Therapeutic Strategies. Int J Mol Sci, 2023. 24(14).
30. Ariga, S., History and Future of HER2-Targeted Therapy for Advanced Gastric Cancer. J Clin Med, 2023. 12(10).
31. Xia, X., et al., The History and Development of HER2 Inhibitors. Pharmaceuticals (Basel), 2023. 16(10).
32. Pegram, M., et al., Evolving perspectives on the treatment of HR+/HER2+ metastatic breast cancer. Ther Adv Med Oncol, 2023. 15: p. 17588359231187201.
33. Riese 2nd, D.J. and V. Dwivedi. A recombinant retroviral expression vector (pLXSN-HygR) based on pLXSN that confers resistance to hygromycin and pLXSN-HygR derivatives that encode EGFR, ERBB2, or ERBB3. protocols.io 2023 [Accessed February 3, 2024]; August 23, 2023:[Available from: https://www.protocols.io/view/a-recombinant-retroviral-expression-vector-plxsn-h-j8nlkooq1v5r/v1.
34. Wilson, K.J., et al., EGFR ligands exhibit functional differences in models of paracrine and autocrine signaling. Growth Factors, 2012. 30(2): p. 107-16.
35. Chang, H., et al., Ligands for ErbB-family receptors encoded by a neuregulin-like gene. Nature, 1997. 387(6632): p. 509-12.
36. Hobbs, S.S., et al., Neuregulin isoforms exhibit distinct patterns of ErbB family receptor activation. Oncogene, 2002. 21(55): p. 8442-52.
37. Hobbs, S.S., et al., Five carboxyl-terminal residues of neuregulin2 are critical for stimulation of signaling by the ErbB4 receptor tyrosine kinase. Oncogene, 2004. 23(4): p. 883-93.
38. Hobbs, S.S., R.M. Gallo, and D.J. Riese, Jr., Phe45 of NRG2beta is critical for the affinity of NRG2beta for ErbB4 and for potent stimulation of ErbB4 signaling by NRG2beta*. Growth Factors, 2005. 23(4): p. 273-83.
39. Wilson, K.J., et al., Inter-conversion of neuregulin2 full and partial agonists for ErbB4. Biochem Biophys Res Commun, 2007. 364(2): p. 351-7.
40. Wilson, K.J., et al., The Q43L mutant of neuregulin 2beta is a pan-ErbB receptor antagonist.Biochem J, 2012. 443(1): p. 133-44.