Accumulation, purification and biological activity of elastin-like polypeptide fusions in plants

A. Kaldis, A. J. Conley, Jussi Joensuu, A. Ahmad, J. E. Brandle, R. Menassa

Research output: Contribution to conferenceConference articleScientific

Abstract

Many therapeutic proteins have been expressed in plants with varying levels of accumulation. Two major challenges hindering the economical production of recombinant proteins include inadequate accumulation levels and the lack of efficient, inexpensive purification methods. Our research addresses these issues by utilizing an elastin-like polypeptide (ELP) as a fusion with various recombinant proteins in tobacco plants or BY-2 cell suspensions. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)n that are valuable for bioseparation, acting as thermally responsive tags for the non-chromatographic purification of recombinant proteins. Our data suggest that smaller ELP tags (n=10-40) result in the highest accumulation levels of their respective fusion partners (up to 5% of TSP for interleukin-10 and over 30% of TSP for GFP), whereas purification is more efficient with larger ELP tags (n=80-120). ELP fusions with 30 pentapeptide repeats provide the best compromise between improved accumulation and effective purification. Interestingly, ELP fusions tagged with GFP appear as large, mobile fluorescent protein bodies using confocal microscopy. These "bodies" may exclude the recombinant protein from normal physiological turnover, and may be responsible for the positive effect on recombinant protein accumulation. The inverse transition cycling property of ELPs has allowed us to develop a quick capture step in the purification of recombinant protein fusions which were subsequently evaluated for biological activity. We found that several recombinant proteins including erythropoietin and an scFv antibody maintained their biological activity when fused to ELP tags. These results and their implications for the production of recombinant proteins in plants will be discussed.
Original languageEnglish
Publication statusPublished - 2009
MoE publication typeNot Eligible
EventThird International Conference on Plant-Based Vaccines and Antibodies - Verona, Italy
Duration: 15 Jun 200917 Jun 2009

Conference

ConferenceThird International Conference on Plant-Based Vaccines and Antibodies
CountryItaly
CityVerona
Period15/06/0917/06/09

Fingerprint

elastin
recombinant proteins
bioactive properties
polypeptides
recombinant fusion proteins
biopharmaceuticals
erythropoietin
purification methods
protein bodies
biopolymers
interleukin-10
cell suspension culture
tobacco
antibodies

Cite this

Kaldis, A., Conley, A. J., Joensuu, J., Ahmad, A., Brandle, J. E., & Menassa, R. (2009). Accumulation, purification and biological activity of elastin-like polypeptide fusions in plants. Paper presented at Third International Conference on Plant-Based Vaccines and Antibodies, Verona, Italy.
Kaldis, A. ; Conley, A. J. ; Joensuu, Jussi ; Ahmad, A. ; Brandle, J. E. ; Menassa, R. / Accumulation, purification and biological activity of elastin-like polypeptide fusions in plants. Paper presented at Third International Conference on Plant-Based Vaccines and Antibodies, Verona, Italy.
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abstract = "Many therapeutic proteins have been expressed in plants with varying levels of accumulation. Two major challenges hindering the economical production of recombinant proteins include inadequate accumulation levels and the lack of efficient, inexpensive purification methods. Our research addresses these issues by utilizing an elastin-like polypeptide (ELP) as a fusion with various recombinant proteins in tobacco plants or BY-2 cell suspensions. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)n that are valuable for bioseparation, acting as thermally responsive tags for the non-chromatographic purification of recombinant proteins. Our data suggest that smaller ELP tags (n=10-40) result in the highest accumulation levels of their respective fusion partners (up to 5{\%} of TSP for interleukin-10 and over 30{\%} of TSP for GFP), whereas purification is more efficient with larger ELP tags (n=80-120). ELP fusions with 30 pentapeptide repeats provide the best compromise between improved accumulation and effective purification. Interestingly, ELP fusions tagged with GFP appear as large, mobile fluorescent protein bodies using confocal microscopy. These {"}bodies{"} may exclude the recombinant protein from normal physiological turnover, and may be responsible for the positive effect on recombinant protein accumulation. The inverse transition cycling property of ELPs has allowed us to develop a quick capture step in the purification of recombinant protein fusions which were subsequently evaluated for biological activity. We found that several recombinant proteins including erythropoietin and an scFv antibody maintained their biological activity when fused to ELP tags. These results and their implications for the production of recombinant proteins in plants will be discussed.",
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Kaldis, A, Conley, AJ, Joensuu, J, Ahmad, A, Brandle, JE & Menassa, R 2009, 'Accumulation, purification and biological activity of elastin-like polypeptide fusions in plants' Paper presented at Third International Conference on Plant-Based Vaccines and Antibodies, Verona, Italy, 15/06/09 - 17/06/09, .

Accumulation, purification and biological activity of elastin-like polypeptide fusions in plants. / Kaldis, A.; Conley, A. J.; Joensuu, Jussi; Ahmad, A.; Brandle, J. E.; Menassa, R.

2009. Paper presented at Third International Conference on Plant-Based Vaccines and Antibodies, Verona, Italy.

Research output: Contribution to conferenceConference articleScientific

TY - CONF

T1 - Accumulation, purification and biological activity of elastin-like polypeptide fusions in plants

AU - Kaldis, A.

AU - Conley, A. J.

AU - Joensuu, Jussi

AU - Ahmad, A.

AU - Brandle, J. E.

AU - Menassa, R.

PY - 2009

Y1 - 2009

N2 - Many therapeutic proteins have been expressed in plants with varying levels of accumulation. Two major challenges hindering the economical production of recombinant proteins include inadequate accumulation levels and the lack of efficient, inexpensive purification methods. Our research addresses these issues by utilizing an elastin-like polypeptide (ELP) as a fusion with various recombinant proteins in tobacco plants or BY-2 cell suspensions. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)n that are valuable for bioseparation, acting as thermally responsive tags for the non-chromatographic purification of recombinant proteins. Our data suggest that smaller ELP tags (n=10-40) result in the highest accumulation levels of their respective fusion partners (up to 5% of TSP for interleukin-10 and over 30% of TSP for GFP), whereas purification is more efficient with larger ELP tags (n=80-120). ELP fusions with 30 pentapeptide repeats provide the best compromise between improved accumulation and effective purification. Interestingly, ELP fusions tagged with GFP appear as large, mobile fluorescent protein bodies using confocal microscopy. These "bodies" may exclude the recombinant protein from normal physiological turnover, and may be responsible for the positive effect on recombinant protein accumulation. The inverse transition cycling property of ELPs has allowed us to develop a quick capture step in the purification of recombinant protein fusions which were subsequently evaluated for biological activity. We found that several recombinant proteins including erythropoietin and an scFv antibody maintained their biological activity when fused to ELP tags. These results and their implications for the production of recombinant proteins in plants will be discussed.

AB - Many therapeutic proteins have been expressed in plants with varying levels of accumulation. Two major challenges hindering the economical production of recombinant proteins include inadequate accumulation levels and the lack of efficient, inexpensive purification methods. Our research addresses these issues by utilizing an elastin-like polypeptide (ELP) as a fusion with various recombinant proteins in tobacco plants or BY-2 cell suspensions. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)n that are valuable for bioseparation, acting as thermally responsive tags for the non-chromatographic purification of recombinant proteins. Our data suggest that smaller ELP tags (n=10-40) result in the highest accumulation levels of their respective fusion partners (up to 5% of TSP for interleukin-10 and over 30% of TSP for GFP), whereas purification is more efficient with larger ELP tags (n=80-120). ELP fusions with 30 pentapeptide repeats provide the best compromise between improved accumulation and effective purification. Interestingly, ELP fusions tagged with GFP appear as large, mobile fluorescent protein bodies using confocal microscopy. These "bodies" may exclude the recombinant protein from normal physiological turnover, and may be responsible for the positive effect on recombinant protein accumulation. The inverse transition cycling property of ELPs has allowed us to develop a quick capture step in the purification of recombinant protein fusions which were subsequently evaluated for biological activity. We found that several recombinant proteins including erythropoietin and an scFv antibody maintained their biological activity when fused to ELP tags. These results and their implications for the production of recombinant proteins in plants will be discussed.

M3 - Conference article

ER -

Kaldis A, Conley AJ, Joensuu J, Ahmad A, Brandle JE, Menassa R. Accumulation, purification and biological activity of elastin-like polypeptide fusions in plants. 2009. Paper presented at Third International Conference on Plant-Based Vaccines and Antibodies, Verona, Italy.