Expression and Purification of G Protein-coupled Receptor from Polycystin-1 Using MBP Fusion in E. coli
Hala Salim Sonbol 1*, Alaa Muqbil Al-sirhani 2
Journal of Angiotherapy 8(6) 1-9 https://doi.org/10.25163/angiotherapy.869691
Submitted: 10 April 2024 Revised: 04 June 2024 Published: 12 June 2024
Abstract
Background: Polycystin-1 (PC1), encoded by the PKD1 gene, is critical in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD) when mutated. PC1 contains a unique proteolytic site, the G protein-coupled receptor (GPS) domain, suggesting a role in G protein signaling regulation. Disruption in the GPS region of PC1 leads to structural reorganization within renal cells, promoting aggressive renal cystogenesis. Understanding the process of GPS cleavage is crucial as it regulates the production and localization of PC1, essential for kidney growth and function. Methods: In this study, we amplified the GPS region of polycystin-1 from genomic DNA and cloned it into the pET-21a(+)-MBP(TEV) expression vector. The molecular chaperone maltose binding protein (MBP) was employed to ensure proper folding of the fusion proteins. The resulting fusion protein, MBP-His-GPS, was expressed in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). Results: The GPS region of PC1 was successfully amplified and cloned into the expression vector. The fusion protein, MBP-His-GPS, exhibited high levels of expression in E. coli. The purification process using IMAC resulted in a high yield of purified fusion protein. The use of MBP as a fusion partner enhanced the solubility and stability of the target protein, facilitating its purification. Conclusion: The study successfully developed a methodology for expressing and purifying the GPS region of PC1, utilizing MBP to improve protein solubility and stability. This technique is essential for the in-depth study and manipulation of PC1 in the laboratory. The approach underlines the potential of MBP fusion in enhancing protein expression and simplifying purification processes, thereby advancing molecular techniques for PC1 research and contributing to a better understanding of its role in ADPKD.
Keywords: Polycystin-1 (PC1), Autosomal dominant polycystic kidney disease (ADPKD), G protein-coupled receptor (GPS), Maltose binding protein (MBP), Protein expression and purification
References
Araç, D., Aust, G., Calebiro, D., Engel, F. B., Formstone, C., Goffinet, A., Hamann, J., Kittel, R. J., Liebscher, I., Lin, H. H., Monk, K. R., Petrenko, A., Piao, X., Prömel, S., Schiöth, H. B., Schwartz, T. W., Stacey, M., Ushkaryov, Y. A., Wobus, M., Wolfrum, U., Xu, L., & Langenhan, T. (2012). Dissecting signaling and functions of adhesion G protein–coupled receptors. Annals of the New York Academy of Sciences, 1276, 1-25. https://doi.org/10.1111/j.1749-6632.2012.06820.x
Bach, H., Mazor, Y., Shaky, S., Shoham-Lev, A., Berdichevsky, Y., Gutnick, D. L., & Benhar, I. (2001). Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies. Journal of Molecular Biology, 312(1), 79-93.
Caroccia, K. E., Estephan, R., Cohen, L. S., Arshava, B., Hauser, M., Zerbe, O., Becker, J. M., & Naider, F. (2011). Expression and biophysical analysis of a triple-transmembrane domain-containing fragment from a yeast G protein-coupled receptor. Biopolymers, 96(6), 757-771. https://doi.org/10.1002/bip.21614
Chapin, H. C., & Caplan, M. J. (2010). The cell biology of polycystic kidney disease. Journal of Cell Biology, 191(4), 701-710.
di Guan, C., Li, P., Riggs, P. D., & Inouye, H. (1988). Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene, 67(1), 21-30. https://doi.org/10.1016/0378-1119(88)90004-2
Feng, S., Streets, A. J., Nesin, V., Tran, U., Nie, H., Onopiuk, M., & Ong, A. C. (2017). The sorting nexin 3 retromer pathway regulates the cell surface localization and activity of a Wnt-activated polycystin channel complex. Journal of the American Society of Nephrology, 28(10), 2973-2984.
Han, Y., Guo, W., Su, B., Guo, Y., Wang, J., Chu, B., & Yang, G. (2018). High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag. Protein Expression and Purification, 142, 25-31.
Kapust, R. B., & Waugh, D. S. (1999). Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Science, 8(8), 1668-1674. https://doi.org/10.1110/ps.8.8.1668
Khan, A., Ning, S., Nestor, J. G., Weng, C., Hripcsak, G. M., Harris, P. C., & Kiryluk, K. (2023). Polygenic risk affects the penetrance of monogenic kidney disease. medRxiv. https://doi.org/10.1101/2023.05.07.23289614
Ki, M. R., & Pack, S. P. (2020). Fusion tags to enhance heterologous protein expression. Applied Microbiology and Biotechnology, 104(6), 2411-2425.
Krasnoperov, V., Lu, Y., Buryanovsky, L., Neubert, T. A., Ichtchenko, K., & Petrenko, A. G. (2002). Post-translational proteolytic processing of the calcium-independent receptor of α-latrotoxin (CIRL), a natural chimera of the cell adhesion protein and the G protein-coupled receptor: role of the G protein-coupled receptor proteolysis site (GPS) motif. Journal of Biological Chemistry, 277(48), 46518-46526.
Kurbegovic, A., Kim, H., Xu, H., Yu, S., Cruanès, J., Maser, R. L., & Qian, F. (2014). Novel functional complexity of polycystin-1 by GPS cleavage in vivo: role in polycystic kidney disease. Molecular and Cellular Biology, 34(17), 3341-3353.
Lea, W. A., McGreal, K., Sharma, M., Parnell, S. C., Zelenchuk, L., Charlesworth, M. C., & Ward, C. J. (2020). Analysis of the polycystin complex (PCC) in human urinary exosome–like vesicles (ELVs). Scientific Reports, 10(1), 1500.
Lin, H. H., Chang, G. W., Davies, J. Q., Stacey, M., Harris, J., & Gordon, S. (2004). Autocatalytic cleavage of the EMR2 receptor occurs at a conserved G protein-coupled receptor proteolytic site motif. Journal of Biological Chemistry, 279(30), 31823-31832.
Malhas, A. N., Abuknesha, R. A., & Price, R. G. (2001). Polycystin-1: immunoaffinity isolation and characterisation by mass spectrometry. FEBS Letters, 505, 313-316.
Maser, R. L., & Calvet, J. (2020). Adhesion GPCRs as a paradigm for understanding polycystin-1 G protein regulation. Cellular Signalling, 72, 109637. https://doi.org/10.1016/j.cellsig.2020.109637
McCusker, E. C., Bane, S. E., O'Malley, M. A., & Robinson, A. S. (2007). Heterologous GPCR expression: a bottleneck to obtaining crystal structures. Biotechnology Progress, 23(3), 540-547. https://doi.org/10.1021/bp060349b
Merrick, D., Mistry, K., Wu, J., Gresko, N., Baggs, J. E., Hogenesch, J. B., & Caplan, M. J. (2019). Polycystin-1 regulates bone development through an interaction with the transcriptional coactivator TAZ. Human Molecular Genetics, 28(1), 16-30.
Mohammadian, N., Mohammadian, H., Moazen, F., Pakdel, M. H., Jahanian-Najafabadi, A., & Sadeghi, H. M. (2020). Optimization of solvent media to solubilize TEV protease using response surface method. Research in Pharmaceutical Sciences, 15(4), 331.
Mullis, K. B., & Faloona, F. A. (1987). Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods in Enzymology, 55, 335-350.
Nims, N., Vassmer, D., & Maser, R. L. (2003). Transmembrane domain analysis of polycystin-1, the product of the polycystic kidney disease-1 (PKD1) gene: Evidence for 11 membrane-spanning domains. Biochemistry, 42(44), 13035-13048. https://doi.org/10.1021/bi035074c
Ong, A. C., & Harris, P. C. (2005). Molecular pathogenesis of ADPKD: the polycystin complex gets complex. Kidney International, 67(4), 1234-1247. https://doi.org/10.1111/j.1523-1755.2005.00201.x
Potetinova, Z., Tantry, S., Cohen, L. S., Caroccia, K. E., Arshava, B., Becker, J. M., & Naider, F. (2012). Large multiple transmembrane domain fragments of a G protein-coupled receptor: biosynthesis, purification, and biophysical studies. Biopolymers, 98(5), 485-500. https://doi.org/10.1002/bip.22122
Qian, F. (2015). The role of G-protein coupled receptor proteolytic site (GPS) cleavage in polycystin-1 biogenesis, trafficking and function. In Li, X. (Ed.), Polycystic Kidney Disease (Chapter 11). Codon Publications. https://doi.org/10.15586/codon.pkd.2015.ch11
Russell, D. W., & Sambrook, J. (2001). Molecular cloning: A laboratory manual (3rd ed., Vol. 1). Cold Spring Harbor Laboratory.
Sandford, R., Sgotto, B., Aparicio, S., Brenner, S., Vaudin, M., & Wilson, R. K. (1997). Comparative analysis of the polycystic kidney disease 1 (PKD1) gene reveals an integral membrane glycoprotein with multiple evolutionary conserved domains. Human Molecular Genetics, 6, 1483–1489. https://doi.org/10.1093/hmg/6.9.1483
Sonbol, H. S., & AlRashidi, A. A. (2022). Cloning and expression of receptor of egg jelly protein of polycystic kidney disease 1 gene in human receptor of egg jelly protein. Pharmacophore, 13(6), 97-105. https://doi.org/10.51847/vqgHaBLLgJ
Sonbol, H. S., & Alsirhani, A. M. (2023). Cloning and expression of recombinant human GPS domain as cytoplasmic N-terminal fragment of polycystin-1. Bioscience Research, 20(1), 38-43.
Su, X., Wu, M., Yao, G., El-Jouni, W., Luo, C., Tabari, A., & Zhou, J. (2015). Regulation of polycystin-1 ciliary trafficking by motifs at its C-terminus and polycystin-2 but not by cleavage at the GPS site. Journal of Cell Science, 128(22), 4063-4073.
Vetrini, F., D’Alessandro, L. C., Akdemir, Z. C., Braxton, A., Azamian, M. S., Eldomery, M. K., & Yang, Y. (2016). Bi-allelic mutations in PKD1L1 are associated with laterality defects in humans. American Journal of Human Genetics, 99(4), 886-893.
Ward, C. J. (1994). The polycystic kidney-disease-1 gene encodes a 14 Kb transcript and lies within a duplicated region on chromosome-16. Cell, 78(4), 725-725.
Waugh, D. S. (2011). An overview of enzymatic reagents for the removal of affinity tags. Protein Expression and Purification, 80(2), 283-293.
Weston, B. S., Bagnéris, C., Price, R. G., & Stirling, J. L. (2001). The polycystin-1 C-type lectin domain binds carbohydrate in a calcium-dependent manner, and interacts with extracellular matrix proteins in vitro. Biochimica et Biophysica Acta, 1536(2-3), 161-176.
Xu, Y., Streets, A. J., Hounslow, A. M., Tran, U., Jean-Alphonse, F., Needham, A. J., Vilardaga, J. P., Wessely, O., Williamson, M. P., & Ong, A. C. (2016). The polycystin-1, lipoxygenase, and α-toxin domain regulates polycystin-1 trafficking. Journal of the American Society of Nephrology, 27(4), 1159-1173. https://doi.org/10.1681/ASN.2014111074
Zhang, D. P., Jing, X. R., Fan, A. W., Liu, H., Nie, Y., & Xu, Y. (2020). Active expression of membrane-bound L-amino acid deaminase from Proteus mirabilis in recombinant Escherichia coli by fusion with maltose-binding protein for enhanced catalytic performance. Catalysts, 10(2), 215.
Zhou, Y., Lu, Z., Wang, X., Selvaraj, J. N., & Zhang, G. (2018). Genetic engineering modification and fermentation optimization for extracellular production of recombinant proteins using Escherichia coli. Applied Microbiology and Biotechnology, 102, 1545-1556.
View Dimensions
View Altmetric
Save
Citation
View
Share