Characterization of recombinant glutelin type-B 5-like protein from proso millet

Felix Akharume; Konstantin K. Korotkov; Akinbode Adedeji

doi:10.13031/aim.202000272

Characterization of recombinant glutelin type-B 5-like protein from proso millet

Felix Akharume, Konstantin K. Korotkov, Akinbode Adedeji

Research output: Contribution to conference › Paper

1 Scopus citations

Abstract

Proso millet glutelin protein fractions make up its second-largest protein mostly used in proso millet concentrates and isolates. In this experiment, we identified the gene of glutelin type-B 5-like protein isoform of the glutelin fraction with over 40% structural identity with the crystal structure of recombinant pro-11S globulin of pumpkin (2E9Q). We further cloned the gene of glutelin type-B 5-like protein with attached six continuous histidine codons into a pET 21d expression vector, overexpressed in rosetta Escherichia coli cells, purified the protein from inclusion bodies by unfolding with 8 M urea and refolding in 0.5 M Arginine with 20 mM CHES (2-(Cyclohexylamino)ethanesulfonic acid). Characterization of purified protein reveals above 90% pure protein based on an SDS-PAGE gel analysis with a band 22.1 kDa. Additionally, the protein revealed a monodispersed particle size distribution with a broad range of particle size (6.50 - 43.82 nm) and a higher-order aggregation with molecular weight range 60-70 kDa.

Original language	English
DOIs	https://doi.org/10.13031/aim.202000272
State	Published - 2020
Event	2020 ASABE Annual International Meeting - Virtual, Online Duration: Jul 13 2020 → Jul 15 2020

Conference

Conference	2020 ASABE Annual International Meeting
City	Virtual, Online
Period	7/13/20 → 7/15/20

Bibliographical note

Funding Information:
This work was supported by the Kentucky Agricultural Experiment Station (KAES), and the National Institute of Food and Agriculture (NIFA), U.S. Department of Agriculture, Hatch-Multistate project #: 1007893. Additionally, authors would like to thank Catherine Chaton, Zamakhaeva Svetlana, Williamson Zachary, and Shakhashiro Muna for their contributions to this project.

Publisher Copyright:
© ASABE 2020 Annual International Meeting.

Funding

This work was supported by the Kentucky Agricultural Experiment Station (KAES), and the National Institute of Food and Agriculture (NIFA), U.S. Department of Agriculture, Hatch-Multistate project #: 1007893. Additionally, authors would like to thank Catherine Chaton, Zamakhaeva Svetlana, Williamson Zachary, and Shakhashiro Muna for their contributions to this project.

Funders	Funder number
U.S. Department of Agriculture	1007893
National Institute of Food and Agriculture
Kentucky Agricultural Experiment Station

Keywords

Proso millet
Protein purification
Recombinant DNA

ASJC Scopus subject areas

Agronomy and Crop Science
Bioengineering

Access to Document

10.13031/aim.202000272

Cite this

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title = "Characterization of recombinant glutelin type-B 5-like protein from proso millet",

abstract = "Proso millet glutelin protein fractions make up its second-largest protein mostly used in proso millet concentrates and isolates. In this experiment, we identified the gene of glutelin type-B 5-like protein isoform of the glutelin fraction with over 40\% structural identity with the crystal structure of recombinant pro-11S globulin of pumpkin (2E9Q). We further cloned the gene of glutelin type-B 5-like protein with attached six continuous histidine codons into a pET 21d expression vector, overexpressed in rosetta Escherichia coli cells, purified the protein from inclusion bodies by unfolding with 8 M urea and refolding in 0.5 M Arginine with 20 mM CHES (2-(Cyclohexylamino)ethanesulfonic acid). Characterization of purified protein reveals above 90\% pure protein based on an SDS-PAGE gel analysis with a band 22.1 kDa. Additionally, the protein revealed a monodispersed particle size distribution with a broad range of particle size (6.50 - 43.82 nm) and a higher-order aggregation with molecular weight range 60-70 kDa.",

keywords = "Proso millet, Protein purification, Recombinant DNA",

author = "Felix Akharume and Korotkov, \{Konstantin K.\} and Akinbode Adedeji",

note = "Funding Information: This work was supported by the Kentucky Agricultural Experiment Station (KAES), and the National Institute of Food and Agriculture (NIFA), U.S. Department of Agriculture, Hatch-Multistate project \#: 1007893. Additionally, authors would like to thank Catherine Chaton, Zamakhaeva Svetlana, Williamson Zachary, and Shakhashiro Muna for their contributions to this project. Publisher Copyright: {\textcopyright} ASABE 2020 Annual International Meeting.; 2020 ASABE Annual International Meeting ; Conference date: 13-07-2020 Through 15-07-2020",

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doi = "10.13031/aim.202000272",

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AU - Akharume, Felix

AU - Korotkov, Konstantin K.

AU - Adedeji, Akinbode

N1 - Funding Information: This work was supported by the Kentucky Agricultural Experiment Station (KAES), and the National Institute of Food and Agriculture (NIFA), U.S. Department of Agriculture, Hatch-Multistate project #: 1007893. Additionally, authors would like to thank Catherine Chaton, Zamakhaeva Svetlana, Williamson Zachary, and Shakhashiro Muna for their contributions to this project. Publisher Copyright: © ASABE 2020 Annual International Meeting.

PY - 2020

Y1 - 2020

N2 - Proso millet glutelin protein fractions make up its second-largest protein mostly used in proso millet concentrates and isolates. In this experiment, we identified the gene of glutelin type-B 5-like protein isoform of the glutelin fraction with over 40% structural identity with the crystal structure of recombinant pro-11S globulin of pumpkin (2E9Q). We further cloned the gene of glutelin type-B 5-like protein with attached six continuous histidine codons into a pET 21d expression vector, overexpressed in rosetta Escherichia coli cells, purified the protein from inclusion bodies by unfolding with 8 M urea and refolding in 0.5 M Arginine with 20 mM CHES (2-(Cyclohexylamino)ethanesulfonic acid). Characterization of purified protein reveals above 90% pure protein based on an SDS-PAGE gel analysis with a band 22.1 kDa. Additionally, the protein revealed a monodispersed particle size distribution with a broad range of particle size (6.50 - 43.82 nm) and a higher-order aggregation with molecular weight range 60-70 kDa.

AB - Proso millet glutelin protein fractions make up its second-largest protein mostly used in proso millet concentrates and isolates. In this experiment, we identified the gene of glutelin type-B 5-like protein isoform of the glutelin fraction with over 40% structural identity with the crystal structure of recombinant pro-11S globulin of pumpkin (2E9Q). We further cloned the gene of glutelin type-B 5-like protein with attached six continuous histidine codons into a pET 21d expression vector, overexpressed in rosetta Escherichia coli cells, purified the protein from inclusion bodies by unfolding with 8 M urea and refolding in 0.5 M Arginine with 20 mM CHES (2-(Cyclohexylamino)ethanesulfonic acid). Characterization of purified protein reveals above 90% pure protein based on an SDS-PAGE gel analysis with a band 22.1 kDa. Additionally, the protein revealed a monodispersed particle size distribution with a broad range of particle size (6.50 - 43.82 nm) and a higher-order aggregation with molecular weight range 60-70 kDa.

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KW - Protein purification

KW - Recombinant DNA

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ER -

Characterization of recombinant glutelin type-B 5-like protein from proso millet

Abstract

Conference

Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

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