Preproricin expressed in Nicotiana tabacum cells in vitro is fully processed and biologically active

Edward P. Tagge, John Chandler, Billie Harris, Mihaly Czako, Laszlo Marton, Mark C. Willingham, Chris Burbage, Lawrence Afrin, Arthur E. Frankel

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Ricin, the highly toxic glycoprotein expressed in the endosperm of castor seeds, is composed of a galactosebinding lectin B chain (RTB) disulfide linked to a RNA N-glycosidase A chain (RTA). Chemically modified ricin has been conjugated to monoclonal antibodies and used for targeted therapy of cancer and autoimmune diseases. Replacement of chemically coupled molecules with a genetically engineered targeted ricin would improve homogeneity and yield and permit structural changes in the fusion toxin to be introduced readily by oligonucleotide-directed mutagenesis. Previous methods of expression of ricin fusion proteins have been limited to expression of RTA or RTB moieties alone or expression of incompletely processed toxin in Xenopus laevis oocytes. In the present study, we introduced the cDNA encoding preproricin into cultured tobacco cells via Agrobacterium tumefaciens- mediated gene transfer. Yields of ricin in soluble cell extracts were 1 μg/g in cells or, approximately, 0.1% of the total soluble protein. The ricin was partially purified by P2 monoclonal antibody anti-RTB affinity chromatography. The RTA and RTB immunoreactive material migrated on SDS-PAGE at 65 kDa under nonreducing conditions and at 32-35 kDa under reducing conditions. The tobacco ricin bound to immobilized asialofetuin as avidly as castor bean ricin, suggesting intact sugar binding. Tobacco ricin inhibited rabbit reticulocyte lysate protein translation similar to castor bean ricin (IC50 of 3 x 10-12 M for tobacco ricin and 1 x 10-11 M for castor bean ricin). The human cutaneous T cell lymphoma cell line HUT102 showed similar sensitivity to tobacco ricin when compared to castor bean ricin (IC50 = 9 x 10-13 and 2 x 10-12 M, respectively). The efficiency of gene transfer, reasonable levels of expression, and full post-translational processing indicate that this expression system is suitable for production of ricin fusion toxins for therapeutic applications.

Original languageEnglish (US)
Pages (from-to)109-118
Number of pages10
JournalProtein Expression and Purification
Volume8
Issue number1
DOIs
StatePublished - Aug 1996

Fingerprint

Ricin
Tobacco
Castor Bean
Rapid thermal annealing
Gene transfer
Fusion reactions
In Vitro Techniques
preproricin
Inhibitory Concentration 50
Monoclonal Antibodies
Ribosome Inactivating Proteins
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Affinity chromatography
Cutaneous T-Cell Lymphoma
Agrobacterium tumefaciens
Mutagenesis
Endosperm
Proteins
T-cells
Poisons

ASJC Scopus subject areas

  • Biochemistry

Cite this

Preproricin expressed in Nicotiana tabacum cells in vitro is fully processed and biologically active. / Tagge, Edward P.; Chandler, John; Harris, Billie; Czako, Mihaly; Marton, Laszlo; Willingham, Mark C.; Burbage, Chris; Afrin, Lawrence; Frankel, Arthur E.

In: Protein Expression and Purification, Vol. 8, No. 1, 08.1996, p. 109-118.

Research output: Contribution to journalArticle

Tagge, EP, Chandler, J, Harris, B, Czako, M, Marton, L, Willingham, MC, Burbage, C, Afrin, L & Frankel, AE 1996, 'Preproricin expressed in Nicotiana tabacum cells in vitro is fully processed and biologically active', Protein Expression and Purification, vol. 8, no. 1, pp. 109-118. https://doi.org/10.1006/prep.1996.0080
Tagge, Edward P. ; Chandler, John ; Harris, Billie ; Czako, Mihaly ; Marton, Laszlo ; Willingham, Mark C. ; Burbage, Chris ; Afrin, Lawrence ; Frankel, Arthur E. / Preproricin expressed in Nicotiana tabacum cells in vitro is fully processed and biologically active. In: Protein Expression and Purification. 1996 ; Vol. 8, No. 1. pp. 109-118.
@article{e6bf0e10013f4b5281f7233e605998ae,
title = "Preproricin expressed in Nicotiana tabacum cells in vitro is fully processed and biologically active",
abstract = "Ricin, the highly toxic glycoprotein expressed in the endosperm of castor seeds, is composed of a galactosebinding lectin B chain (RTB) disulfide linked to a RNA N-glycosidase A chain (RTA). Chemically modified ricin has been conjugated to monoclonal antibodies and used for targeted therapy of cancer and autoimmune diseases. Replacement of chemically coupled molecules with a genetically engineered targeted ricin would improve homogeneity and yield and permit structural changes in the fusion toxin to be introduced readily by oligonucleotide-directed mutagenesis. Previous methods of expression of ricin fusion proteins have been limited to expression of RTA or RTB moieties alone or expression of incompletely processed toxin in Xenopus laevis oocytes. In the present study, we introduced the cDNA encoding preproricin into cultured tobacco cells via Agrobacterium tumefaciens- mediated gene transfer. Yields of ricin in soluble cell extracts were 1 μg/g in cells or, approximately, 0.1{\%} of the total soluble protein. The ricin was partially purified by P2 monoclonal antibody anti-RTB affinity chromatography. The RTA and RTB immunoreactive material migrated on SDS-PAGE at 65 kDa under nonreducing conditions and at 32-35 kDa under reducing conditions. The tobacco ricin bound to immobilized asialofetuin as avidly as castor bean ricin, suggesting intact sugar binding. Tobacco ricin inhibited rabbit reticulocyte lysate protein translation similar to castor bean ricin (IC50 of 3 x 10-12 M for tobacco ricin and 1 x 10-11 M for castor bean ricin). The human cutaneous T cell lymphoma cell line HUT102 showed similar sensitivity to tobacco ricin when compared to castor bean ricin (IC50 = 9 x 10-13 and 2 x 10-12 M, respectively). The efficiency of gene transfer, reasonable levels of expression, and full post-translational processing indicate that this expression system is suitable for production of ricin fusion toxins for therapeutic applications.",
author = "Tagge, {Edward P.} and John Chandler and Billie Harris and Mihaly Czako and Laszlo Marton and Willingham, {Mark C.} and Chris Burbage and Lawrence Afrin and Frankel, {Arthur E.}",
year = "1996",
month = "8",
doi = "10.1006/prep.1996.0080",
language = "English (US)",
volume = "8",
pages = "109--118",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Preproricin expressed in Nicotiana tabacum cells in vitro is fully processed and biologically active

AU - Tagge, Edward P.

AU - Chandler, John

AU - Harris, Billie

AU - Czako, Mihaly

AU - Marton, Laszlo

AU - Willingham, Mark C.

AU - Burbage, Chris

AU - Afrin, Lawrence

AU - Frankel, Arthur E.

PY - 1996/8

Y1 - 1996/8

N2 - Ricin, the highly toxic glycoprotein expressed in the endosperm of castor seeds, is composed of a galactosebinding lectin B chain (RTB) disulfide linked to a RNA N-glycosidase A chain (RTA). Chemically modified ricin has been conjugated to monoclonal antibodies and used for targeted therapy of cancer and autoimmune diseases. Replacement of chemically coupled molecules with a genetically engineered targeted ricin would improve homogeneity and yield and permit structural changes in the fusion toxin to be introduced readily by oligonucleotide-directed mutagenesis. Previous methods of expression of ricin fusion proteins have been limited to expression of RTA or RTB moieties alone or expression of incompletely processed toxin in Xenopus laevis oocytes. In the present study, we introduced the cDNA encoding preproricin into cultured tobacco cells via Agrobacterium tumefaciens- mediated gene transfer. Yields of ricin in soluble cell extracts were 1 μg/g in cells or, approximately, 0.1% of the total soluble protein. The ricin was partially purified by P2 monoclonal antibody anti-RTB affinity chromatography. The RTA and RTB immunoreactive material migrated on SDS-PAGE at 65 kDa under nonreducing conditions and at 32-35 kDa under reducing conditions. The tobacco ricin bound to immobilized asialofetuin as avidly as castor bean ricin, suggesting intact sugar binding. Tobacco ricin inhibited rabbit reticulocyte lysate protein translation similar to castor bean ricin (IC50 of 3 x 10-12 M for tobacco ricin and 1 x 10-11 M for castor bean ricin). The human cutaneous T cell lymphoma cell line HUT102 showed similar sensitivity to tobacco ricin when compared to castor bean ricin (IC50 = 9 x 10-13 and 2 x 10-12 M, respectively). The efficiency of gene transfer, reasonable levels of expression, and full post-translational processing indicate that this expression system is suitable for production of ricin fusion toxins for therapeutic applications.

AB - Ricin, the highly toxic glycoprotein expressed in the endosperm of castor seeds, is composed of a galactosebinding lectin B chain (RTB) disulfide linked to a RNA N-glycosidase A chain (RTA). Chemically modified ricin has been conjugated to monoclonal antibodies and used for targeted therapy of cancer and autoimmune diseases. Replacement of chemically coupled molecules with a genetically engineered targeted ricin would improve homogeneity and yield and permit structural changes in the fusion toxin to be introduced readily by oligonucleotide-directed mutagenesis. Previous methods of expression of ricin fusion proteins have been limited to expression of RTA or RTB moieties alone or expression of incompletely processed toxin in Xenopus laevis oocytes. In the present study, we introduced the cDNA encoding preproricin into cultured tobacco cells via Agrobacterium tumefaciens- mediated gene transfer. Yields of ricin in soluble cell extracts were 1 μg/g in cells or, approximately, 0.1% of the total soluble protein. The ricin was partially purified by P2 monoclonal antibody anti-RTB affinity chromatography. The RTA and RTB immunoreactive material migrated on SDS-PAGE at 65 kDa under nonreducing conditions and at 32-35 kDa under reducing conditions. The tobacco ricin bound to immobilized asialofetuin as avidly as castor bean ricin, suggesting intact sugar binding. Tobacco ricin inhibited rabbit reticulocyte lysate protein translation similar to castor bean ricin (IC50 of 3 x 10-12 M for tobacco ricin and 1 x 10-11 M for castor bean ricin). The human cutaneous T cell lymphoma cell line HUT102 showed similar sensitivity to tobacco ricin when compared to castor bean ricin (IC50 = 9 x 10-13 and 2 x 10-12 M, respectively). The efficiency of gene transfer, reasonable levels of expression, and full post-translational processing indicate that this expression system is suitable for production of ricin fusion toxins for therapeutic applications.

UR - http://www.scopus.com/inward/record.url?scp=0030221377&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030221377&partnerID=8YFLogxK

U2 - 10.1006/prep.1996.0080

DO - 10.1006/prep.1996.0080

M3 - Article

C2 - 8812841

AN - SCOPUS:0030221377

VL - 8

SP - 109

EP - 118

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 1

ER -