Login Register Cart 0
Generic placeholder image

Protein & Peptide Letters

Editor-in-Chief

ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Expression of Brazzein, a Small Sweet-Tasting Protein in Saccharomyces cerevisiae: An Introduction for Production of Sweet Yeasts

Author(s): Akram Kazemi-Nasab and Azar Shahpiri*

Volume 27, Issue 10, 2020

Page: [945 - 952] Pages: 8

DOI: 10.2174/0929866527666200331134431

Price: $65

Abstract

Background: The replacement of carbohydrate sweeteners with protein sweeteners from plants has attracted the interest of researchers because these proteins don’t trigger the insulin response and are more nutritive for consumption in food. Brazzein (Braz) is a small and heat- stable sweet protein that has been originally derived from African plant Pentadiplandra brazzeana. In the present work the solubility, sweetness and yield of recombinant forms of Braz in two expression hosts, E. coli and S. cerevisiae were comprised.

Methods: The codon-optimized gene of Braz was cloned in expression vectors pET28a and pET41a and GPD. The resulted vectors pET28a-Braz and pEt41a-Braz were transformed into Escherichia coli strain Rosetta (DE3) and the vector GPD-Braz was transformd to S. cerevisiae. The expression of Braz in different systems was analyzed by SDS-PAGE and western blotting.

Results: The results verified the heterologous expression of Braz in S. cerevisiae carrying GPDBraz. Also the expression of Braz as carboxy-terminal extensions of His-tag and Glutathione-STransferase (GST) were verified in transgenic E. coli containing pET28a-Braz and pET41a-Braz, respectively.

Conclusion: Although the yield of GST-Braz was higher than His-Braz and Braz expressed in S. cerevisiae, but the higher solubility, sweetness, safety (GRAS) are important advantages of the use of S. cerevisiae as expression host for production of Braz. Therefore the result of present work opens new insights for providing the new sweet yeasts that can be used as food additives.

Keywords: Brazzein, E. coli, recombinant, Saccharomyces cerevisiae, solubility sweetness, glutathione-S-transferase.

« Previous Next »
Graphical Abstract

[1]
Clemens, R.A.; Jones, J.M.; Kern, M.; Lee, S.Y.; Mayhew, E.J.; Slavin, J.L.; Zivanovic, S. Functionality of sugars in foods and health. Compr. Rev. Food Sci. Food Saf., 2016, 15, 443-470.
[http://dx.doi.org/10.1111/1541-4337.12194]
[2]
Kant, R. Sweet proteins-potential replacement for artificial low calorie sweeteners. Nutr. J., 2005, 4, 5.
[http://dx.doi.org/10.1186/1475-2891-4-5] [PMID: 15703077]
[3]
Ming, D.; Hellekant, G. Brazzein, a new high-potency thermostable sweet protein from Pentadiplandra brazzeana B. FEBS Lett., 1994, 355(1), 106-108.
[http://dx.doi.org/10.1016/0014-5793(94)01184-2] [PMID: 7957951]
[4]
Morris, J.A.; Cagan, R.H. Purification of monellin, the sweet principle of Dioscoreophyllum cumminsii. Biochim. Biophys. Acta, 1972, 261(1), 114-122.
[http://dx.doi.org/10.1016/0304-4165(72)90320-0] [PMID: 5012458]
[5]
van der Wel, H.; Loeve, K. Isolation and characterization of thaumatin I and II, the sweet-tasting proteins from Thaumatococcus daniellii Benth. Eur. J. Biochem., 1972, 31(2), 221-225.
[http://dx.doi.org/10.1111/j.1432-1033.1972.tb02522.x] [PMID: 4647176]
[6]
Wel, H.V.D.; Larson, G.; Hladik, A.; Hladik, C.M.; Hellekant, G.; Glaser, D. Isolation and characterization of pentadin, the sweet principle of Pentadiplandra brazzeana baillon. Chem. Senses, 1989, 14, 75-79.
[http://dx.doi.org/10.1093/chemse/14.1.75]
[7]
Liu, X.; Maeda, S.; Hu, Z.; Aiuchi, T.; Nakaya, K.; Kurihara, Y. Purification, complete amino acid sequence and structural characterization of the heat-stable sweet protein, mabinlin II. Eur. J. Biochem., 1993, 211(1-2), 281-287.
[http://dx.doi.org/10.1111/j.1432-1033.1993.tb19896.x] [PMID: 8425538]
[8]
Yamashita, H.; Theerasilp, S.; Aiuchi, T.; Nakaya, K.; Nakamura, Y.; Kurihara, Y. Purification and complete amino acid sequence of a new type of sweet protein taste-modifying activity, curculin. J. Biol. Chem., 1990, 265(26), 15770-15775.
[PMID: 2394746]
[9]
Faus, I. Recent developments in the characterization and biotechnological production of sweet-tasting proteins. Appl. Microbiol. Biotechnol., 2000, 53(2), 145-151.
[http://dx.doi.org/10.1007/s002530050001] [PMID: 10709975]
[10]
Izawa, H.; Ota, M.; Kohmura, M.; Ariyoshi, Y. Synthesis and characterization of the sweet protein brazzein. Biopolymers, 1996, 39(1), 95-101.
[http://dx.doi.org/10.1002/(SICI)1097-0282(199607)39:1<95:AID-BIP10>3.0.CO;2-B] [PMID: 8924630]
[11]
Assadi-Porter, F.M.; Aceti, D.J.; Markley, J.L. Sweetness determinant sites of brazzein, a small, heat-stable, sweet-tasting protein. Arch. Biochem. Biophys., 2000, 376(2), 259-265.
[http://dx.doi.org/10.1006/abbi.2000.1726] [PMID: 10775411]
[12]
Reghelin, E. Plant derived Sweet and Ice Structuring proteins as new tools in food processing. PhD Thesis, Università Degli Studi Di Padova: Itlay, 2012.
[13]
Yun, C.R.; Kong, J.N.; Chung, J.H.; Kim, M.C.; Kong, K.H. Improved secretory production of the sweet-tasting protein, brazzein, in Kluyveromyces lactis. J. Agric. Food Chem., 2016, 64(32), 6312-6316.
[http://dx.doi.org/10.1021/acs.jafc.6b02446] [PMID: 27465609]
[14]
Assadi-Porter, F.M.; Patry, S.; Markley, J.L. Efficient and rapid protein expression and purification of small high disulfide containing sweet protein brazzein in E. coli. Protein Expr. Purif., 2008, 58(2), 263-268.
[http://dx.doi.org/10.1016/j.pep.2007.11.009] [PMID: 18221889]
[15]
Assadi-Porter, F.M.; Aceti, D.J.; Cheng, H.; Markley, J.L. Efficient production of recombinant brazzein, a small, heat-stable, sweet-tasting protein of plant origin. Arch. Biochem. Biophys., 2000, 376(2), 252-258.
[http://dx.doi.org/10.1006/abbi.2000.1725] [PMID: 10775410]
[16]
Lee, J.J.; Kong, J.N.; Do, H.D.; Jo, D.H.; Kong, K.H. Design and efficient soluble expression of a sweet protein, brazzein and minor-form mutant. B. Kor. Chem. Soc., 2010, 31, 3830-3833.
[http://dx.doi.org/10.5012/bkcs.2010.31.12.3830]
[17]
Borodina, I.; Nielsen, J. Advances in metabolic engineering of yeast Saccharomyces cerevisiae for production of chemicals. Biotechnol. J., 2014, 9(5), 609-620.
[http://dx.doi.org/10.1002/biot.201300445] [PMID: 24677744]
[18]
Bekatorou, A.; Psarianos, C.; Koutinas, A.A. Production of food grade yeasts. Food Technol. Biotechnol., 2006, 44, 407-415.
[19]
Kalmus, P.; Orro, T.; Waldmann, A.; Lindjärv, R.; Kask, K. Effect of yeast culture on milk production and metabolic and reproductive performance of early lactation dairy cows. Acta Vet. Scand., 2009, 51, 32-51.
[http://dx.doi.org/10.1186/1751-0147-51-32] [PMID: 19650887]
[20]
Berlec, A.; Jevnikar, Z.; Majhenič, A.Č.; Rogelj, I.; Štrukelj, B. Expression of the sweet-tasting plant protein brazzein in Escherichia coli and Lactococcus lactis: A path toward sweet lactic acid bacteria. Appl. Microbiol. Biotechnol., 2006, 73(1), 158-165.
[http://dx.doi.org/10.1007/s00253-006-0438-y] [PMID: 16703320]
[21]
Mansouri, F.; Modarressi, M.H.; Abolhassani, M.; Parivar, K. Synthesis and production of sweet-tasting protein in E. coli and purification by amylose resin. J. Sci. Islam. Repub. Iran, 2011, 22, 105-110.
[22]
Sivashanmugam, A.; Murray, V.; Cui, C.; Zhang, Y.; Wang, J.; Li, Q. Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Sci., 2009, 18(5), 936-948.
[http://dx.doi.org/10.1002/pro.102] [PMID: 19384993]
[23]
Sambrook, J.; Fritsch, E.F.; Maniatis, T. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press: New York, NY, 1989.
[24]
Gietz, R.D.; Woods, R.A. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol., 2002, 350, 87-96.
[http://dx.doi.org/10.1016/S0076-6879(02)50957-5] [PMID: 12073338]
[25]
Zhang, T.; Lei, J.; Yang, H.; Xu, K.; Wang, R.; Zhang, Z. An improved method for whole protein extraction from yeast Saccharomyces cerevisiae. Yeast, 2011, 28(11), 795-798.
[http://dx.doi.org/10.1002/yea.1905] [PMID: 21972073]
[26]
Jiang, S.; Liu, S.; Zhao, C.; Wu, C. Developing protocols of tricine-SDS-PAGE for separation of polypeptides in the mass range 1-30 kDa with minigel electrophoresis system. Int. J. Electrochem. Sci., 2016, 11, 640-649.
[27]
Schägger, H. Tricine-SDS-PAGE. Nat. Protoc., 2006, 1(1), 16-22.
[http://dx.doi.org/10.1038/nprot.2006.4] [PMID: 17406207]
[28]
Schägger, H.; von Jagow, G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem., 1987, 166(2), 368-379.
[http://dx.doi.org/10.1016/0003-2697(87)90587-2] [PMID: 2449095]
[29]
Jo, H.J.; Noh, J.S.; Kong, K.H. Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis. Protein Expr. Purif., 2013, 90(2), 84-89.
[http://dx.doi.org/10.1016/j.pep.2013.05.001] [PMID: 23684772]
[30]
Lamphear, B.J.; Barker, D.K.; Brooks, C.A.; Delaney, D.E.; Lane, J.R.; Beifuss, K.; Love, R.; Thompson, K.; Mayor, J.; Clough, R.; Harkey, R.; Poage, M.; Drees, C.; Horn, M.E.; Streatfield, S.J.; Nikolov, Z.; Woodard, S.L.; Hood, E.E.; Jilka, J.M.; Howard, J.A. Expression of the sweet protein brazzein in maize for production of a new commercial sweetener. Plant Biotechnol. J., 2005, 3(1), 103-114.
[http://dx.doi.org/10.1111/j.1467-7652.2004.00105.x] [PMID: 17168903]
[31]
Moure, V.R.; Razzera, G.; Araújo, L.M.; Oliveira, M.A.S.; Gerhardt, E.C.M.; Müller-Santos, M.; Almeida, F.; Pedrosa, F.O.; Valente, A.P.; Souza, E.M.; Huergo, L.F. Heat stability of Proteobacterial PII protein facilitate purification using a single chromatography step. Protein Expr. Purif., 2012, 81(1), 83-88.
[http://dx.doi.org/10.1016/j.pep.2011.09.008] [PMID: 21963770]
[32]
Yan, S.; Song, H.; Pang, D.; Zou, Q.; Li, L.; Yan, Q.; Fan, N.; Zhao, X.; Yu, H.; Li, Z.; Wang, H.; Gao, F.; Ouyang, H.; Lai, L. Expression of plant sweet protein brazzein in the milk of transgenic mice. PLoS One, 2013, 8(10), e76769.
[http://dx.doi.org/10.1371/journal.pone.0076769] [PMID: 24155905]
[33]
Caldwell, J.E.; Abildgaard, F.; Džakula, Z.; Ming, D.; Hellekant, G.; Markley, J.L. Solution structure of the thermostable sweet-tasting protein brazzein. Nat. Struct. Biol., 1998, 5(6), 427-431.
[http://dx.doi.org/10.1038/nsb0698-427] [PMID: 9628478]
[34]
Poirier, N.; Roudnitzky, N.; Brockhoff, A.; Belloir, C.; Maison, M.; Thomas-Danguin, T.; Meyerhof, W.; Briand, L. Efficient production and characterization of the sweet-tasting brazzein secreted by the yeast Pichia pastoris. J. Agric. Food Chem., 2012, 60(39), 9807-9814.
[http://dx.doi.org/10.1021/jf301600m] [PMID: 22958103]

Rights & Permissions Print Cite
Article Metrics
24
1
© 2024 Bentham Science Publishers | Privacy Policy