Partial recovery of MRJP1 protein expressed in Pichia pastoris using chromatographic techniques
Keywords:
MRJP1, Pichia pastoris, ion exchange chromatography, size exclusion chromatography
Abstract
Major Royal Jelly Protein 1 (MRJP1) is the main protein component of the bee-produced complex mixture royal jelly, which is the only nutrient source for queen bees promoting increased lifespan, body size and fertility. Recombinant production of MRJP1 represents an alternative to direct extraction from royal jelly. Production in Pichia pastoris results in high density biomass, with a supernatant containing high amount of impurities. Various methods have been applied to recover and/or purify MRJP1. Here, exploiting the physicochemical properties of MRJP1, reverse phase chromatography (RPC), size exclusion chromatography (SEC) and ion-exchange chromatography (IEX) were investigated as alternative methods to recover MRJP1 directly from supernatant. All techniques showed a 57-kDa band in SDS-PAGE analysis, corresponding to the size of recombinant MRJP1, with contaminants attributed to culture media. However, SEC coupled to IEX evidenced a single peak in the chromatogram corresponding to MRJP1 which suggest it may be a good protocol to recover recombinant MRJP1 from P. pastoris supernatant. This approach serves as a procedure to identify MRJP1 in fermentation culture of P. pastoris. This is the first report about characterization of IEX-based recovery of recombinant Apis mellifera MRJP1 produced in Pichia pastoris without the use of histidine tags.
References
Acikara, Ö.B. (2013). Ion-Exchange Chromatography and Its Applications, Column Chromatography, Dean F. Martin and Barbara B. Martin, IntechOpen, DOI: 10.5772/55744. Available from: https://www.intechopen.com/books/column-chromatography/ion-exchange-chromatography-and-its-applications. Accessed: February 12, 2020.
Buttstedt, A., Moritz, R.F.A. and Erler, S. (2014). Origin and function of the major royal jelly proteins of the honeybee ( Apis mellifera ) as members of the yellow gene family. Biol. Rev. 49, 255–269. https://doi.org/10.1111/brv.12052
Carta, G. and Jungbauer, A. (2010). Protein Chromatography: Process Development and Scale-Up, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Chen, D., Xin, X.X., Qian, H.C., Yu, Z.Y. and Shen, L.R. (2016). Evaluation of the major royal jelly proteins as an alternative to fetal bovine serum in culturing human cell lines. J Zhejiang Univ-Sci B (Biomed & Biotechnol). 17(6):476-483. 10.1631/jzus.B1500295
Cruz, G.C.N., Garcia, L., Silva, A.J., Barbosa, J.A.R.G., Ricart, C.A.O., Freitas, S.M. and Sousa, M.V. (2011). Calcium effect and pH-dependence on self-association and structural stability of the Apis mellifera major royal jelly protein 1. Apidologie. 42, 252–269. https://doi.org/10.1007/s13592-011-0025-9.
Detienne, G., De Haes, W., Ernst, U.R., Schoofs, L. and Temmerman, L. (2014). Royalactin extends lifespan of Caenorhabditis elegans through epidermal growth factor signaling, EXG. 60, 129–135. https://doi.org/10.1016/j.exger.2014.09.021.
Dong, Y., Zhang, F., Wang, Z., Du, L., Hao, A., Jiang, B., Tian, M., Li, Q., Jia, Q., Wang, S. and Xiu, Z. (2012). Extraction and purification of recombinant human serum albumin from Pichia pastoris broths using aqueous two-phase system combined with hydrophobic interaction chromatography, J Chromatogr A 1245:143–149. 10.1016/j.chroma.2012.05.041
Furusawa, T., Arai, Y., Kato, K. and Ichihara, K. (2016). Quantitative Analysis of Apisin, a Major Protein Unique to Royal Jelly, Evidence-Based Complement. Altern. Med. 2016, 1–9. https://doi.org/10.1155/2016/5040528.
GE Healthcare Life Sciences (2020). Fundamentals of size exclusion chromatography. https://www.gelifesciences.com/en/us/solutions/protein-research/knowledge-center/protein-purification-methods/size-exclusion-chromatography. Accessed: February 13, 2020.
GE Healthcare. (2004). Strategies for protein purification. Handbook 28-9833-31, Uppsala, Sweden.
GE Healthcare. (2006). Hydrophobic Interaction and Reversed Phase Chromatography. Principles and Methods. Handbook 11-0012-69, Uppsala, Sweden.
González-González, M., Mayolo-Deloisa, K. and Rito-Palomares, M. (2012). PEGylation, detection and chromatographic purification of site-specific PEGylated CD133-Biotin antibody in route to stem cell separation. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 893–894, 182–186. https://doi.org/10.1016/j.jchromb.2012.03.002.
Ibarra-Herrera, C.C., Torres-Acosta, M.A., Mendoza-Ochoa, G.I., Aguilar-Yañez, J.M. and Rito-Palomares, M. (2014). Recovery of major royal jelly protein 1 expressed in Pichia pastoris in aqueous two-phase systems. J. Chem. Technol. Biotechnol. 89, 941–947. https://doi.org/10.1002/jctb.4342.
Júdová, J., Šutka, R., Klaudiny, J., Lišková, D., Ow, D.W., and Šimúth, J. (2004). Transformation of tobacco plants with cDNA encoding honeybee royal jelly MRJP1. Biologia Plantarum, 48, 185–191. https://doi.org/10.1023/B:BIOP.0000033443.60872.f1
Kamakuraa, M., Fukuda, T., Fukushima, M. and Yonekura, M. (2001). Storage-dependent Degradation of 57-kDa Protein in Royal Jelly: a Possible Marker for Freshness. Biosci. Biotechnol. Biochem. 65, 277–284. https://doi.org/10.1271/bbb.65.277.
Kamakurab, M., Suenobu, N. and Fukushima, M. (2001). Fifty-seven-kDa Protein in Royal Jelly Enhances Proliferation of Primary Cultured Rat Hepatocytes and Increases Albumin Production in the Absence of Serum. Biochem Biophys Res Commun. 874, 865–874. https://doi.org/10.1006/bbrc.2001.4656.
Kamakurac, M. and Sakaki, T. (2006). A hypopharyngeal gland protein of the worker honeybee Apis mellifera L. enhances proliferation of primary-cultured rat hepatocytes and suppresses apoptosis in the absence of serum. Protein Expr. Purif. 45, 307–314. https://doi.org/10.1016/j.pep.2005.08.004.
Kamakurad, M. (2011). Royalactin induces queen differentiation in honeybees. Nature. 473, 478-483. doi:10.1038/nature10093
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227(5259):680-5. 10.1038/227680a0.
Li, S.W., Sun, Y., Donelan, W., Yu, H., Scian, J., Tang, D., and Yang, L.J. (2010). Expression, purification, and characterization of recombinant human pancreatic duodenal homeobox-1 protein in Pichia pastoris. Protein Expr Purif. 72(2):157–161. 10.1016/j.pep.2010.04.004
Maccani, A., Landes, N., Stadlmayr, G., Maresch, D., Leitner, C., Maurer, M., Gasser, B., Ernst, W., Kunert, R. and Mattanovich, D. (2014). Pichia pastoris secretes recombinant proteins less efficiently than Chinese hamster ovary cells but allows higher space-time yields for less complex proteins. Biotechnol J. 9(4):526-37. 10.1002/biot.201300305
Mandacaru, S.C., do Vale, L.H.F., Vahidi, S., Xiao, Y., Skinner, O.S., Ricart, C.A.O., Kelleher, N.L., Valle de Sousa, M. and Konermann, L. (2017). Characterizing the Structure and Oligomerization of Major Royal Jelly Protein 1 (MRJP1) by Mass Spectrometry and Complementary Biophysical Tools. Biochemistry. 56(11), 1645–1655. 10.1021/acs.biochem.7b00020.
Musa, M., Mohamad-Nasir, N.F. and Thirumulu, K.P. (2014). Evaluation of Royal Jelly as an alternative to fetal bovine serum in cell culture. Afr J Tradit Complement Altern Med. 11, 148–155. http://dx.doi.org/10.4314/ajtcam.v11i1.23.
Neuhoff, V., Arold, N., Taube, D. and Ehrhardt, W. (1988). Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis. 9(6):255-62. 10.1002/elps.1150090603
Pasupuleti, V.R., Sammugam, L., Ramesh, N. and Gan, S.H. (2017). Review Article Honey, Propolis, and Royal Jelly: A Comprehensive Review of Their Biological Actions and Health Benefits. Oxid Med Cell Longev. 2017. https://doi.org/10.1155/2017/1259510.
Shen, L., Wang, Y., Zhai,, L. and Zhou, W. (2015). Determination of royal jelly freshness by ELISA with a highly specific anti-apalbumin 1, major royal jelly protein 1 antibody *. J Zhejiang Univ-Sci B (Biomed & Biotechnol). 16, 155–166. https://doi.org/10.1631/jzus.B1400223.
Shen, L., Zhang, W., Jin, F., Zhang, L., Chen, Z., Liu, L., Parnell, L.D., Lai, C-Q and LI, D. (2010). Expression of Recombinant AccMRJP1 Protein from Royal Jelly of Chinese Honeybee in Pichia pastoris and Its Proliferation Activity in an Insect Cell Line. J. Agric. Food Chem. 2010, 9190–9197. https://doi.org/10.1021/jf1007133.
Shorter, J.R., Geisz, M., Özsoy, E. and Magwire, M.M. (2015). The Effects of Royal Jelly on Fitness Traits and Gene Expression in Drosophila melanogaster. PLoS ONE 10 (7), 1–10. https://doi.org/10.1371/journal.pone.0134612.
Wan, D.C., Morgan, S.L., Spencley, A.L., Mariano, N., Chang, E.Y., Shankar, G., Luo, Y., Li, T.H., Huh, D., Huynh, S.K., Garcia, J.M., Dovey, C.M., Lumb, J., Liu, L., Brown, K.V., Bermudez, A., Luong, R., Zeng, H., Mascetti, V.L., Pitteri, S.J., Wang, J., Tu, H., Quarta, M., Sebastiano, V., Nusse, R., Rando, T.A., Carette, J.E., Bazan, J.F. and Wang, K.C. (2018). Honey bee Royalactin unlocks conserved pluripotency pathway in mammals. Nat. Commun. 9. https://doi.org/10.1038/s41467-018-06256-4.
Tian, W., Li, M., Guo, H., Peng, W., Xue, X., Hu, Y., Liu, Y., Zhao, Y., Fang, X., Wang, K., Li, X., Tong, Y., Conlon, M., Wu, W., Ren, F. and Chen, Z. (2018). Architecture of the native major royal jelly protein 1 oligomer. Nature Communications, 9:3373. doi: 10.1038/s41467-018-05619-1
Xin, X.X., Chen, Y., Chen, D., Xiao, F., Parnell, L.D., Zhao, J., Liu, L., Ordovas, J.M., Lai, C.Q. and Shen, L.R. (2016). Supplementation with Major Royal-Jelly Proteins Increases Lifespan, Feeding, and Fecundity in Drosophila. J Agric Food Chem. 64(29):5803-12. doi: 10.1021/acs.jafc.6b00514.
Buttstedt, A., Moritz, R.F.A. and Erler, S. (2014). Origin and function of the major royal jelly proteins of the honeybee ( Apis mellifera ) as members of the yellow gene family. Biol. Rev. 49, 255–269. https://doi.org/10.1111/brv.12052
Carta, G. and Jungbauer, A. (2010). Protein Chromatography: Process Development and Scale-Up, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Chen, D., Xin, X.X., Qian, H.C., Yu, Z.Y. and Shen, L.R. (2016). Evaluation of the major royal jelly proteins as an alternative to fetal bovine serum in culturing human cell lines. J Zhejiang Univ-Sci B (Biomed & Biotechnol). 17(6):476-483. 10.1631/jzus.B1500295
Cruz, G.C.N., Garcia, L., Silva, A.J., Barbosa, J.A.R.G., Ricart, C.A.O., Freitas, S.M. and Sousa, M.V. (2011). Calcium effect and pH-dependence on self-association and structural stability of the Apis mellifera major royal jelly protein 1. Apidologie. 42, 252–269. https://doi.org/10.1007/s13592-011-0025-9.
Detienne, G., De Haes, W., Ernst, U.R., Schoofs, L. and Temmerman, L. (2014). Royalactin extends lifespan of Caenorhabditis elegans through epidermal growth factor signaling, EXG. 60, 129–135. https://doi.org/10.1016/j.exger.2014.09.021.
Dong, Y., Zhang, F., Wang, Z., Du, L., Hao, A., Jiang, B., Tian, M., Li, Q., Jia, Q., Wang, S. and Xiu, Z. (2012). Extraction and purification of recombinant human serum albumin from Pichia pastoris broths using aqueous two-phase system combined with hydrophobic interaction chromatography, J Chromatogr A 1245:143–149. 10.1016/j.chroma.2012.05.041
Furusawa, T., Arai, Y., Kato, K. and Ichihara, K. (2016). Quantitative Analysis of Apisin, a Major Protein Unique to Royal Jelly, Evidence-Based Complement. Altern. Med. 2016, 1–9. https://doi.org/10.1155/2016/5040528.
GE Healthcare Life Sciences (2020). Fundamentals of size exclusion chromatography. https://www.gelifesciences.com/en/us/solutions/protein-research/knowledge-center/protein-purification-methods/size-exclusion-chromatography. Accessed: February 13, 2020.
GE Healthcare. (2004). Strategies for protein purification. Handbook 28-9833-31, Uppsala, Sweden.
GE Healthcare. (2006). Hydrophobic Interaction and Reversed Phase Chromatography. Principles and Methods. Handbook 11-0012-69, Uppsala, Sweden.
González-González, M., Mayolo-Deloisa, K. and Rito-Palomares, M. (2012). PEGylation, detection and chromatographic purification of site-specific PEGylated CD133-Biotin antibody in route to stem cell separation. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 893–894, 182–186. https://doi.org/10.1016/j.jchromb.2012.03.002.
Ibarra-Herrera, C.C., Torres-Acosta, M.A., Mendoza-Ochoa, G.I., Aguilar-Yañez, J.M. and Rito-Palomares, M. (2014). Recovery of major royal jelly protein 1 expressed in Pichia pastoris in aqueous two-phase systems. J. Chem. Technol. Biotechnol. 89, 941–947. https://doi.org/10.1002/jctb.4342.
Júdová, J., Šutka, R., Klaudiny, J., Lišková, D., Ow, D.W., and Šimúth, J. (2004). Transformation of tobacco plants with cDNA encoding honeybee royal jelly MRJP1. Biologia Plantarum, 48, 185–191. https://doi.org/10.1023/B:BIOP.0000033443.60872.f1
Kamakuraa, M., Fukuda, T., Fukushima, M. and Yonekura, M. (2001). Storage-dependent Degradation of 57-kDa Protein in Royal Jelly: a Possible Marker for Freshness. Biosci. Biotechnol. Biochem. 65, 277–284. https://doi.org/10.1271/bbb.65.277.
Kamakurab, M., Suenobu, N. and Fukushima, M. (2001). Fifty-seven-kDa Protein in Royal Jelly Enhances Proliferation of Primary Cultured Rat Hepatocytes and Increases Albumin Production in the Absence of Serum. Biochem Biophys Res Commun. 874, 865–874. https://doi.org/10.1006/bbrc.2001.4656.
Kamakurac, M. and Sakaki, T. (2006). A hypopharyngeal gland protein of the worker honeybee Apis mellifera L. enhances proliferation of primary-cultured rat hepatocytes and suppresses apoptosis in the absence of serum. Protein Expr. Purif. 45, 307–314. https://doi.org/10.1016/j.pep.2005.08.004.
Kamakurad, M. (2011). Royalactin induces queen differentiation in honeybees. Nature. 473, 478-483. doi:10.1038/nature10093
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227(5259):680-5. 10.1038/227680a0.
Li, S.W., Sun, Y., Donelan, W., Yu, H., Scian, J., Tang, D., and Yang, L.J. (2010). Expression, purification, and characterization of recombinant human pancreatic duodenal homeobox-1 protein in Pichia pastoris. Protein Expr Purif. 72(2):157–161. 10.1016/j.pep.2010.04.004
Maccani, A., Landes, N., Stadlmayr, G., Maresch, D., Leitner, C., Maurer, M., Gasser, B., Ernst, W., Kunert, R. and Mattanovich, D. (2014). Pichia pastoris secretes recombinant proteins less efficiently than Chinese hamster ovary cells but allows higher space-time yields for less complex proteins. Biotechnol J. 9(4):526-37. 10.1002/biot.201300305
Mandacaru, S.C., do Vale, L.H.F., Vahidi, S., Xiao, Y., Skinner, O.S., Ricart, C.A.O., Kelleher, N.L., Valle de Sousa, M. and Konermann, L. (2017). Characterizing the Structure and Oligomerization of Major Royal Jelly Protein 1 (MRJP1) by Mass Spectrometry and Complementary Biophysical Tools. Biochemistry. 56(11), 1645–1655. 10.1021/acs.biochem.7b00020.
Musa, M., Mohamad-Nasir, N.F. and Thirumulu, K.P. (2014). Evaluation of Royal Jelly as an alternative to fetal bovine serum in cell culture. Afr J Tradit Complement Altern Med. 11, 148–155. http://dx.doi.org/10.4314/ajtcam.v11i1.23.
Neuhoff, V., Arold, N., Taube, D. and Ehrhardt, W. (1988). Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis. 9(6):255-62. 10.1002/elps.1150090603
Pasupuleti, V.R., Sammugam, L., Ramesh, N. and Gan, S.H. (2017). Review Article Honey, Propolis, and Royal Jelly: A Comprehensive Review of Their Biological Actions and Health Benefits. Oxid Med Cell Longev. 2017. https://doi.org/10.1155/2017/1259510.
Shen, L., Wang, Y., Zhai,, L. and Zhou, W. (2015). Determination of royal jelly freshness by ELISA with a highly specific anti-apalbumin 1, major royal jelly protein 1 antibody *. J Zhejiang Univ-Sci B (Biomed & Biotechnol). 16, 155–166. https://doi.org/10.1631/jzus.B1400223.
Shen, L., Zhang, W., Jin, F., Zhang, L., Chen, Z., Liu, L., Parnell, L.D., Lai, C-Q and LI, D. (2010). Expression of Recombinant AccMRJP1 Protein from Royal Jelly of Chinese Honeybee in Pichia pastoris and Its Proliferation Activity in an Insect Cell Line. J. Agric. Food Chem. 2010, 9190–9197. https://doi.org/10.1021/jf1007133.
Shorter, J.R., Geisz, M., Özsoy, E. and Magwire, M.M. (2015). The Effects of Royal Jelly on Fitness Traits and Gene Expression in Drosophila melanogaster. PLoS ONE 10 (7), 1–10. https://doi.org/10.1371/journal.pone.0134612.
Wan, D.C., Morgan, S.L., Spencley, A.L., Mariano, N., Chang, E.Y., Shankar, G., Luo, Y., Li, T.H., Huh, D., Huynh, S.K., Garcia, J.M., Dovey, C.M., Lumb, J., Liu, L., Brown, K.V., Bermudez, A., Luong, R., Zeng, H., Mascetti, V.L., Pitteri, S.J., Wang, J., Tu, H., Quarta, M., Sebastiano, V., Nusse, R., Rando, T.A., Carette, J.E., Bazan, J.F. and Wang, K.C. (2018). Honey bee Royalactin unlocks conserved pluripotency pathway in mammals. Nat. Commun. 9. https://doi.org/10.1038/s41467-018-06256-4.
Tian, W., Li, M., Guo, H., Peng, W., Xue, X., Hu, Y., Liu, Y., Zhao, Y., Fang, X., Wang, K., Li, X., Tong, Y., Conlon, M., Wu, W., Ren, F. and Chen, Z. (2018). Architecture of the native major royal jelly protein 1 oligomer. Nature Communications, 9:3373. doi: 10.1038/s41467-018-05619-1
Xin, X.X., Chen, Y., Chen, D., Xiao, F., Parnell, L.D., Zhao, J., Liu, L., Ordovas, J.M., Lai, C.Q. and Shen, L.R. (2016). Supplementation with Major Royal-Jelly Proteins Increases Lifespan, Feeding, and Fecundity in Drosophila. J Agric Food Chem. 64(29):5803-12. doi: 10.1021/acs.jafc.6b00514.
Published
2020-08-03
How to Cite
Robles-Zamora, A., Enriquez-Ochoa, D., Ureña-Herrera, M., Aguilar-Yañez, J., Brunck, M., & Mayolo-Deloisa, K. (2020). Partial recovery of MRJP1 protein expressed in Pichia pastoris using chromatographic techniques. Revista Mexicana De Ingeniería Química, 20(1), 147-160. https://doi.org/10.24275/rmiq/Bio1713
Section
Biotechnology
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