Development of the Method to Produce Functionally Active Recombinant Streptavidin in Escherichia coli Cells
Streptavidin is a homotetrameric protein produced by Streptomyces avidinii, each subunit of which binds biotin (vitamin H), forming a stable complex (Kd = 10-15 М). Streptavidin-biotin coreaction is widely used in analytical systems, for targeted delivery of compounds, for affinity purification, etc...
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| Format: | Article |
| Language: | English |
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Siberian Federal University
2020-06-01
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| Series: | Журнал Сибирского федерального университета: Серия Биология |
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| Online Access: | http://elib.sfu-kras.ru/bitstream/2311/135353/1/08_Bashmakova.pdf |
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| author | Eugenia E. Bashmakova Alexander N. Kudryavtsev Ludmila A. Frank |
| author_facet | Eugenia E. Bashmakova Alexander N. Kudryavtsev Ludmila A. Frank |
| author_sort | Eugenia E. Bashmakova |
| collection | DOAJ |
| description | Streptavidin is a homotetrameric protein produced by Streptomyces avidinii, each subunit of which binds biotin (vitamin H), forming a stable complex (Kd = 10-15 М). Streptavidin-biotin coreaction is widely used in analytical systems, for targeted delivery of compounds, for affinity purification, etc. The aim of this study was to develop a rational technique to produce functionally active recombinant streptavidin. Recombinant Escherichia coli strains producing minimal core and full-sized streptavidin variants were obtained. The E. coli BL21 Codon Plus (DE3) RIPL, as host cells, and the pET19b plasmid carrying gene of minimally-sized core (miniSAV) or full-sized (SAV) streptavidin were used. Synthesis of miniSAV results in its localization as insoluble inclusion bodies. Denatured miniSAV yield was 130 mg per liter of E. coli c ulture. T he r enaturation g ives o nly 10- 15 % of the functionally active protein. Full-sized streptavidin localizes in the cytoplasm in a soluble state, but its toxicity causes low yield of the protein (10-13 mg per liter of the culture). The induction of SAV synthesis at the end of the logarithmic stage of cell growth was found to increase the yield of SAV approximately 2-fold. The yield of functionally active protein was 30 mg per liter culture. SAV was produced practically in individual state after affine chromatography on 2-iminobiotin agarose. One molecule of full-sized streptavidin bound 3.9 biotin molecules as was shown by colorimetric analysis using HABA (4-hydroxyazobenzene-2-carboxylic acid). Both streptavidins form sandwichtype complexes with biotinylated molecules in solid-phase microassay conditions. E. coli BL21 Codon Plus (DE3) RIPL/pET19bSAV strain was stable during storage with 20 % glycerol at -70 °С, which was shown by repeated two-year reseeding. The streptavidin producing strain (E. coli BL21 Codon Plus (DE3) RIPL/pET19bSAV) is deposited in the Collection for extremophile microorganisms and type cultures (Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk), No. 3505. The method for producing functionally active recombinant streptavidin developed in this study ensures its availability for biotechnological research |
| first_indexed | 2024-12-21T18:09:30Z |
| format | Article |
| id | doaj.art-699dade4b86b4e42a2ae5a7995bdd22a |
| institution | Directory Open Access Journal |
| issn | 1997-1389 2313-5530 |
| language | English |
| last_indexed | 2024-12-21T18:09:30Z |
| publishDate | 2020-06-01 |
| publisher | Siberian Federal University |
| record_format | Article |
| series | Журнал Сибирского федерального университета: Серия Биология |
| spelling | doaj.art-699dade4b86b4e42a2ae5a7995bdd22a2022-12-21T18:54:49ZengSiberian Federal UniversityЖурнал Сибирского федерального университета: Серия Биология1997-13892313-55302020-06-0113221822910.17516/1997-1389-0324http://elib.sfu-kras.ru/handle/2311/135353Development of the Method to Produce Functionally Active Recombinant Streptavidin in Escherichia coli CellsEugenia E. Bashmakova0Alexander N. Kudryavtsev1Ludmila A. Frank2Institute of Biophysics SB RAS FRC «Krasnoyarsk Science Center SB RAS» Krasnoyarsk, Russian FederationInstitute of Biophysics SB RAS FRC «Krasnoyarsk Science Center SB RAS» Krasnoyarsk, Russian FederationInstitute of Biophysics SB RAS FRC «Krasnoyarsk Science Center SB RAS» Krasnoyarsk, Russian FederationStreptavidin is a homotetrameric protein produced by Streptomyces avidinii, each subunit of which binds biotin (vitamin H), forming a stable complex (Kd = 10-15 М). Streptavidin-biotin coreaction is widely used in analytical systems, for targeted delivery of compounds, for affinity purification, etc. The aim of this study was to develop a rational technique to produce functionally active recombinant streptavidin. Recombinant Escherichia coli strains producing minimal core and full-sized streptavidin variants were obtained. The E. coli BL21 Codon Plus (DE3) RIPL, as host cells, and the pET19b plasmid carrying gene of minimally-sized core (miniSAV) or full-sized (SAV) streptavidin were used. Synthesis of miniSAV results in its localization as insoluble inclusion bodies. Denatured miniSAV yield was 130 mg per liter of E. coli c ulture. T he r enaturation g ives o nly 10- 15 % of the functionally active protein. Full-sized streptavidin localizes in the cytoplasm in a soluble state, but its toxicity causes low yield of the protein (10-13 mg per liter of the culture). The induction of SAV synthesis at the end of the logarithmic stage of cell growth was found to increase the yield of SAV approximately 2-fold. The yield of functionally active protein was 30 mg per liter culture. SAV was produced practically in individual state after affine chromatography on 2-iminobiotin agarose. One molecule of full-sized streptavidin bound 3.9 biotin molecules as was shown by colorimetric analysis using HABA (4-hydroxyazobenzene-2-carboxylic acid). Both streptavidins form sandwichtype complexes with biotinylated molecules in solid-phase microassay conditions. E. coli BL21 Codon Plus (DE3) RIPL/pET19bSAV strain was stable during storage with 20 % glycerol at -70 °С, which was shown by repeated two-year reseeding. The streptavidin producing strain (E. coli BL21 Codon Plus (DE3) RIPL/pET19bSAV) is deposited in the Collection for extremophile microorganisms and type cultures (Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk), No. 3505. The method for producing functionally active recombinant streptavidin developed in this study ensures its availability for biotechnological researchhttp://elib.sfu-kras.ru/bitstream/2311/135353/1/08_Bashmakova.pdfrecombinant streptavidine. coli protein-producing strainmicroanalysis |
| spellingShingle | Eugenia E. Bashmakova Alexander N. Kudryavtsev Ludmila A. Frank Development of the Method to Produce Functionally Active Recombinant Streptavidin in Escherichia coli Cells Журнал Сибирского федерального университета: Серия Биология recombinant streptavidin e. coli protein-producing strain microanalysis |
| title | Development of the Method to Produce Functionally Active Recombinant Streptavidin in Escherichia coli Cells |
| title_full | Development of the Method to Produce Functionally Active Recombinant Streptavidin in Escherichia coli Cells |
| title_fullStr | Development of the Method to Produce Functionally Active Recombinant Streptavidin in Escherichia coli Cells |
| title_full_unstemmed | Development of the Method to Produce Functionally Active Recombinant Streptavidin in Escherichia coli Cells |
| title_short | Development of the Method to Produce Functionally Active Recombinant Streptavidin in Escherichia coli Cells |
| title_sort | development of the method to produce functionally active recombinant streptavidin in escherichia coli cells |
| topic | recombinant streptavidin e. coli protein-producing strain microanalysis |
| url | http://elib.sfu-kras.ru/bitstream/2311/135353/1/08_Bashmakova.pdf |
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