Jeffrey Staub - Academia.edu (original) (raw)

Papers by Jeffrey Staub

Genome Biology

Large quantities of biologically active human somatotropin have been successfully produced in pla... more Large quantities of biologically active human somatotropin have been successfully produced in plants, highlighting the promise of plant biotechnology for producing pharmaceuticals. Significance and context One of the most potentially useful applications of plant biotechnology is the production of xenogenic (foreign) proteins. The ability to scale-up production easily by planting additional acreage and the avoidance of disease contaminants from animal and bacterial systems are two of the most attractive potential benefits. Previous work in the field has concentrated on vaccine and antibody production, and on the synthesis of industrial enzymes. Staub et al. now demonstrate the in planta production of biologically active human somatotropin (hST), the primary use for which in medicine is the treatment of hypopituitary dwarfism in children. Key results The production of xenogenic proteins in plants requires a number of optimization steps: choosing the right promoter, finding the correct codon usage, targeting the protein to the proper subcellular compartment, blocking potential degradation, and ensuring proper post-translational modification. Problems with any of these steps can lead to little or no protein production, or to synthesis of a biologically inactive protein. Staub et al. had to overcome several of these problems. Initial expression attempts using traditional transgenesis into the nuclear genome resulted in little or no hST production, so the authors instead used transformation of chloroplasts. Transformation of the plastid genome has several advantages. First, a plant cell can contain up to 10,000 plastid genomes, greatly increasing the possibility of high expression of the transgene. Staub et al. report that hST expression in one of the transgenic lines made up 7% of total soluble protein. Second, plastid transformation is via homologous recombination. The hST construct was cloned into a vector containing a selectable marker and portions of the plastid genome. This enabled target-specific insertion of the transgene between the trnV gene and the rps7/3'-rps12operon in the plastid genome. This ensures that there are no positional effects for different constructs. Finally, the plastid genome is not transmitted through pollen, allowing greater biological containment of transgenic crops.

Nature biotechnology, 2000

Transgenic plants have become attractive systems for production of human therapeutic proteins bec... more Transgenic plants have become attractive systems for production of human therapeutic proteins because of the reduced risk of mammalian viral contaminants, the ability to do large scale-up at low cost, and the low maintenance requirements. Here we report a feasibility study for production of a human therapeutic protein through transplastomic transformation technology, which has the additional advantage of increased biological containment by apparent elimination of the transmission of transgenes through pollen. We show that chloroplasts can express a secretory protein, human somatotropin, in a soluble, biologically active, disulfide-bonded form. High concentrations of recombinant protein accumulation are observed (>7% total soluble protein), more than 300-fold higher than a similar gene expressed using a nuclear transgenic approach. The plastid-expressed somatotropin is nearly devoid of complex post-translational modifications, effectively increasing the amount of usable recombinan...

PloS one, 2016

Nitrogen availability is crucial for crop yield with nitrogen fertilizer accounting for a large p... more Nitrogen availability is crucial for crop yield with nitrogen fertilizer accounting for a large percentage of farmers' expenses. However, an untimely or excessive application of fertilizer can increase risks of negative environmental effects. These factors, along with the environmental and energy costs of synthesizing nitrogen fertilizer, led us to seek out novel biotechnology-driven approaches to supply nitrogen to plants. The strategy we focused on involves transgenic expression of nitrogenase, a bacterial multi-subunit enzyme that can capture atmospheric nitrogen. Here we report expression of the active Fe subunit of nitrogenase via integration into the tobacco plastid genome of bacterial gene sequences modified for expression in plastid. Our study suggests that it will be possible to engineer plants that are able to produce their own nitrogen fertilizer by expressing nitrogenase genes in plant plastids.

Biochemical and Biophysical Research Communications, Dec 1, 1990

We have used a prokaryotic promoter-identification vector, pKO-1, to isolate rat mitochondrial DN... more We have used a prokaryotic promoter-identification vector, pKO-1, to isolate rat mitochondrial DNA (mtDNA) sequences that can act as bacterial transcription promoters. Three putative promoter-containing clones that hydridized to mtDNA probes were identified. The strength of the promoters was quantitated by measuring galactokinase activity. The three promoters mapped to three distinct regions of the mtDNA - one within the 5' half of the 16S rRNA gene, one within the ATPase subunit 6 gene, and the last at the carboxy terminal end of the cytochrome oxidase subunit I gene.

Homologous Recombination and Gene Silencing in Plants, 1994

ABSTRACT

Methods in molecular biology (Clifton, N.J.), 2014

Chloroplast transformation has been extremely valuable for the study of plastid biology and gene ... more Chloroplast transformation has been extremely valuable for the study of plastid biology and gene expression, but the tissue culture methodology involved can be laborious, and it can take several months to obtain homoplasmic regenerated plants useful for molecular or physiological studies. In contrast, transformation of tobacco suspension cell plastids provides an easy and efficient system to rapidly evaluate the efficacy of multiple constructs prior to plant regeneration. Suspension cell cultures can be initiated from many cell types, and once established, can be maintained by subculture for more than a year with no loss of transformation efficiency. Using antibiotic selection, homoplasmy is readily achieved in uniform cell colonies useful for comparative gene expression analyses, with the added flexibility to subsequently regenerate plants for in planta studies. Plastids from suspension cells grown in the dark are similar in size and cellular morphology to those in embryogenic cult...

Genome Biology

Large quantities of biologically active human somatotropin have been successfully produced in pla... more Large quantities of biologically active human somatotropin have been successfully produced in plants, highlighting the promise of plant biotechnology for producing pharmaceuticals. Significance and context One of the most potentially useful applications of plant biotechnology is the production of xenogenic (foreign) proteins. The ability to scale-up production easily by planting additional acreage and the avoidance of disease contaminants from animal and bacterial systems are two of the most attractive potential benefits. Previous work in the field has concentrated on vaccine and antibody production, and on the synthesis of industrial enzymes. Staub et al. now demonstrate the in planta production of biologically active human somatotropin (hST), the primary use for which in medicine is the treatment of hypopituitary dwarfism in children. Key results The production of xenogenic proteins in plants requires a number of optimization steps: choosing the right promoter, finding the correct codon usage, targeting the protein to the proper subcellular compartment, blocking potential degradation, and ensuring proper post-translational modification. Problems with any of these steps can lead to little or no protein production, or to synthesis of a biologically inactive protein. Staub et al. had to overcome several of these problems. Initial expression attempts using traditional transgenesis into the nuclear genome resulted in little or no hST production, so the authors instead used transformation of chloroplasts. Transformation of the plastid genome has several advantages. First, a plant cell can contain up to 10,000 plastid genomes, greatly increasing the possibility of high expression of the transgene. Staub et al. report that hST expression in one of the transgenic lines made up 7% of total soluble protein. Second, plastid transformation is via homologous recombination. The hST construct was cloned into a vector containing a selectable marker and portions of the plastid genome. This enabled target-specific insertion of the transgene between the trnV gene and the rps7/3'-rps12operon in the plastid genome. This ensures that there are no positional effects for different constructs. Finally, the plastid genome is not transmitted through pollen, allowing greater biological containment of transgenic crops.

Nature biotechnology, 2000

Transgenic plants have become attractive systems for production of human therapeutic proteins bec... more Transgenic plants have become attractive systems for production of human therapeutic proteins because of the reduced risk of mammalian viral contaminants, the ability to do large scale-up at low cost, and the low maintenance requirements. Here we report a feasibility study for production of a human therapeutic protein through transplastomic transformation technology, which has the additional advantage of increased biological containment by apparent elimination of the transmission of transgenes through pollen. We show that chloroplasts can express a secretory protein, human somatotropin, in a soluble, biologically active, disulfide-bonded form. High concentrations of recombinant protein accumulation are observed (>7% total soluble protein), more than 300-fold higher than a similar gene expressed using a nuclear transgenic approach. The plastid-expressed somatotropin is nearly devoid of complex post-translational modifications, effectively increasing the amount of usable recombinan...

PloS one, 2016

Nitrogen availability is crucial for crop yield with nitrogen fertilizer accounting for a large p... more Nitrogen availability is crucial for crop yield with nitrogen fertilizer accounting for a large percentage of farmers' expenses. However, an untimely or excessive application of fertilizer can increase risks of negative environmental effects. These factors, along with the environmental and energy costs of synthesizing nitrogen fertilizer, led us to seek out novel biotechnology-driven approaches to supply nitrogen to plants. The strategy we focused on involves transgenic expression of nitrogenase, a bacterial multi-subunit enzyme that can capture atmospheric nitrogen. Here we report expression of the active Fe subunit of nitrogenase via integration into the tobacco plastid genome of bacterial gene sequences modified for expression in plastid. Our study suggests that it will be possible to engineer plants that are able to produce their own nitrogen fertilizer by expressing nitrogenase genes in plant plastids.

Biochemical and Biophysical Research Communications, Dec 1, 1990

We have used a prokaryotic promoter-identification vector, pKO-1, to isolate rat mitochondrial DN... more We have used a prokaryotic promoter-identification vector, pKO-1, to isolate rat mitochondrial DNA (mtDNA) sequences that can act as bacterial transcription promoters. Three putative promoter-containing clones that hydridized to mtDNA probes were identified. The strength of the promoters was quantitated by measuring galactokinase activity. The three promoters mapped to three distinct regions of the mtDNA - one within the 5' half of the 16S rRNA gene, one within the ATPase subunit 6 gene, and the last at the carboxy terminal end of the cytochrome oxidase subunit I gene.

Homologous Recombination and Gene Silencing in Plants, 1994

ABSTRACT

Methods in molecular biology (Clifton, N.J.), 2014

Chloroplast transformation has been extremely valuable for the study of plastid biology and gene ... more Chloroplast transformation has been extremely valuable for the study of plastid biology and gene expression, but the tissue culture methodology involved can be laborious, and it can take several months to obtain homoplasmic regenerated plants useful for molecular or physiological studies. In contrast, transformation of tobacco suspension cell plastids provides an easy and efficient system to rapidly evaluate the efficacy of multiple constructs prior to plant regeneration. Suspension cell cultures can be initiated from many cell types, and once established, can be maintained by subculture for more than a year with no loss of transformation efficiency. Using antibiotic selection, homoplasmy is readily achieved in uniform cell colonies useful for comparative gene expression analyses, with the added flexibility to subsequently regenerate plants for in planta studies. Plastids from suspension cells grown in the dark are similar in size and cellular morphology to those in embryogenic cult...