Several major costs associated with the production of biopharmaceuticals or vaccines in fermentation-based systems could be minimized by using plant chloroplasts as bioreactors, which facilitates rapid scale-up. Oral delivery of chloroplast-derived therapeutic proteins through plant cells eliminates expensive purification steps, low temperature storage, transportation and sterile injections for their delivery. Chloroplast transformation technology (CTT) has also been successfully used to engineer valuable agronomic traits and for the production of industrial enzymes and biomaterials. Here, we provide a detailed protocol for the construction of chloroplast expression and integration vectors, selection and regeneration of transformants, evaluation of transgene integration and inheritance, confirmation of transgene expression and extraction, and quantitation and purification of foreign proteins. Integration of appropriate transgenes into chloroplast genomes and the resulting high levels of functional protein expression can be achieved in approximately 6 months in lettuce and tobacco. CTT is eco-friendly because transgenes are maternally inherited in most crop plants.

The currently available human vaccine for anthrax, derived from the culture supernatant of Bacillus anthracis, contains the protective antigen (PA) and traces of the lethal and edema factors, which may contribute to adverse side effects associated with this vaccine. Therefore, an effective expression system that can provide a clean, safe, and efficacious vaccine is required. In an effort to produce anthrax vaccine in large quantities and free of extraneous bacterial contaminants, PA was expressed in transgenic tobacco chloroplasts by inserting the pagA gene into the chloroplast genome. Chloroplast integration of the pagA gene was confirmed by PCR and Southern analysis. Mature leaves grown under continuous illumination contained PA as up to 14.2% of the total soluble protein. Cytotoxicity measurements in macrophage lysis assays showed that chloroplast-derived PA was equal in potency to PA produced in B. anthracis. Subcutaneous immunization of mice with partially purified chloroplast-derived or B. anthracis-derived PA with adjuvant yielded immunoglobulin G titers up to 1:320,000, and both groups of mice survived (100%) challenge with lethal doses of toxin. An average yield of about 150 mg of PA per plant should produce 360 million doses of a purified vaccine free of bacterial toxins edema factor and lethal factor from 1 acre of land. Such high expression levels without using fermenters and the immunoprotection offered by the chloroplast-derived PA should facilitate development of a cleaner and safer anthrax vaccine at a lower production cost. These results demonstrate the immunogenic and immunoprotective properties of plant-derived anthrax vaccine antigen.

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. Keywords transmucosal carrier; furin; ganglioside receptors; genetically modified crops One of the most challenging problems of human health management is the high cost of prescription drugs in developed countries and their lack of availability in developing countries. For example, interferon (IFN) alpha 2b is used for the treatment of viral diseases such as hepatitis C, as well as for certain cancers. However, IFN treatment for four months costs $26,000 in the United States, where more than forty-five million Americans do not have health insurance (1). Several hundred million people in developing countries are infected with hepatitis, but the daily income of one-third of the world population is less than $2 per day (1). The high cost of prescription drugs is due to a number reasons, including fermentation-based production (each fermenter costs several hundred million dollars to build), expensive purification and in vitro processing methods (such as column chromatography, disulfide bond formation) (2), the need for storage and transportation at low temperature and delivery via sterile injections requiring the involvement of hospitals NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript and highly qualified health professionals (1). Therefore, new approaches to minimize or eliminate most of these expenses are urgently needed. Transgenic plants offer many advantages, including the feasibility of the oral delivery of foreign proteins, low cost of production, storage and transportation, heat stability and protection through bioencapsulation, elimination of the need for expensive purification, in ...

The Centers for Disease Control (CDC) lists Bacillus anthracis as a category A agent and estimates the cost of an anthrax attack to exceed US$ 26 billion per 100,000 exposed individuals. Concerns regarding anthrax vaccine purity, a requirement for multiple injections, and a limited supply of the protective antigen (PA), underscore the urgent need for an improved vaccine. Therefore, the 83 kDa immunogenic Bacillus anthracis protective antigen was expressed in transgenic tobacco chloroplasts. The PA gene (pag) was cloned into a chloroplast vector along with the psbA regulatory signals to enhance translation. Chloroplast integration of the transgenes was confirmed by PCR and Southern blot analyses. Crude plant extracts contained up to 2.5 mg full length PA/g of fresh leaf tissue and this showed exceptional stability for several months in stored leaves or crude extracts. Maximum levels of expression were observed in mature leaves under continuous illumination. Co-expression of the ORF2 chaperonin from Bacillus thuringiensis did not increase PA accumulation or induce folding into cuboidal crystals in transgenic chloroplasts. Trypsin, chymotrypsin and furin proteolytic cleavage sites present in PA were protected in transgenic chloroplasts because only full length PA 83 was observed without any degradation products. Both CHAPS and SDS detergents extracted PA with equal efficiency and PA was observed in the soluble fraction. Chloroplast-derived PA was functionally active in lysing mouse macrophages when combined with lethal factor (LF). Crude leaf extracts contained up to 25 microg functional PA/ml. With an average yield of 172 mg of PA per plant using an experimental transgenic cultivar grown in a greenhouse, 400 million doses of vaccine (free of contaminants) could be produced per acre, a yield that could be further enhanced 18-fold using a commercial cultivar in the field.

OBJECTIVE-The key pancreatic transcription factor pancreatic duodenal homeobox-1 (Pdx1), known to control development and maintenance of pancreatic ␤-cells, possesses a protein transduction domain (PTD) that facilitates its entry into cells. We therefore sought to evaluate the capacity of in vivo-administered recombinant Pdx1 (rPdx1) to ameliorate hyperglycemia in mice with streptozotocin-induced diabetes.RESEARCH DESIGN AND METHODS-Cell entry and transcriptional regulatory properties of rPdx1 protein and its PTDdeletion mutant rPdx1⌬ protein, as well as a PTD-green fluorescent protein, were evaluated in vitro. After intraperitoneal rPdx1 injection into mice with streptozotocin-induced diabetes, we assessed its action on blood glucose levels, insulin content, intraperitoneal glucose tolerance test (IPGTT), Pdx1 distribution, pancreatic gene expression, islet cell proliferation, and organ histology.RESULTS-Restoration of euglycemia in Pdx1-treated diabetic mice was evident by improved IPGTT and glucose-stimulated insulin release. Insulin, glucagon, and Ki67 immunostaining revealed increased islet cell number and proliferation in pancreata of rPdx1-treated mice. Real-time PCR of pancreas and liver demonstrated upregulation of INS and PDX1 genes and other genes relevant to pancreas regeneration. While the time course of ␤-cell gene expression and serum/tissue insulin levels indicated that both liver-and pancreas-derived insulin contributed to restoration of normoglycemia, near-total pancreatectomy resulted in hyperglycemia, suggesting that ␤-cell regeneration played the primary role in rPdx1-induced glucose homeostasis.CONCLUSIONS-rPdx1 treatment of mice with streptozotocininduced diabetes promotes ␤-cell regeneration and liver cell reprogramming, leading to restoration of normoglycemia. This novel PTD-based protein therapy offers a promising way to treat patients with diabetes while avoiding potential side effects associated with the use of viral vectors. Diabetes 57:757-769, 2008