Title

Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system

Authors

Authors

A. Limaye; V. Koya; M. Samsam;H. Daniell

Comments

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Abbreviated Journal Title

Faseb J.

Keywords

transmucosal carrier; furin; ganglioside receptors; genetically modified; crops; HEAT-LABILE ENTEROTOXIN; TOXIN B-SUBUNIT; CHOLERA-TOXIN; CRYSTAL-STRUCTURE; PROPROTEIN CONVERTASE; PROTECTIVE ANTIGEN; ESCHERICHIA-COLI; GOLGI-APPARATUS; DIABETIC MICE; CELL-SURFACE; Biochemistry & Molecular Biology; Biology; Cell Biology

Abstract

Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM(1) binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins.

Journal Title

Faseb Journal

Volume

20

Issue/Number

7

Publication Date

1-1-2006

Document Type

Article

Language

English

First Page

959

Last Page

+

WOS Identifier

WOS:000240157700021

ISSN

0892-6638

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