Characterization and Oral Delivery of Proinsulin-Transferrin Fusion Protein Expressed Using ExpressTec.

Proinsulin-transferrin fusion protein (ProINS-Tf) has been designed and successfully expressed from the mammalian HEK293 cells (HEK-ProINS-Tf). It was found that HEK-ProINS-Tf could be converted into an activated form in the liver. Furthermore, HEK-ProINS-Tf was demonstrated as an extra-long acting insulin analogue with liver-specific insulin action in streptozotocin (STZ)-induced type 1 diabetic mice. However, due to the low production yield from transfected HEK293 cells, there are other interesting features, including the oral bioavailability, which have not been fully explored and characterized. To improve the protein production yield, an alternative protein expression system, ExpressTec using transgenic rice (Oryza sativa L.), was used. The intact and active rice-derived ProINS-Tf (ExpressTec-ProINS-Tf) was successfully expressed from the transgenic rice expression system. Our results suggested that, although the insulin-like bioactivity of ExpressTec-ProINS-Tf was slightly lower in vitro, its potency of in vivo blood glucose control was considerably stronger than that of HEK-ProINS-Tf. The oral delivery studies in type 1 diabetic mice demonstrated a prolonged control of blood glucose to near-normal levels after oral administration of ExpressTec-ProINS-Tf. Results in this report suggest that ExpressTec-ProINS-Tf is a promising insulin analog with advantages including low cost, prolonged and liver targeting effects, and most importantly, oral bioactivity.

Human leukemia inhibitory factor produced by the ExpressTec method from rice (Oryza sativa L.) is active in human neural stem cells and mouse induced pluripotent stem cells.

Stem cell-based therapy has the potential to treat an array of human diseases. However, to study the therapeutic potential and safety of these cells, a scalable cell culture medium is needed that is free of human or bovine-derived serum proteins. Thus, cost-effective recombinant serum proteins and cytokines are needed to produce such mediums. One such cytokine, leukemia inhibitory factor (LIF), has been shown to be a critical paracrine factor that maintains stem cell pluripotency in murine embryonic stem cells and human naïve stem cells while simultaneously inhibiting differentiation. We recently produced recombinant human LIF (rhLIF) in a rice-based protein expression system known as ExpressTec. (12) We described expression of rice-derived rhLIF and demonstrated its biological equivalency to E. coli-derived rhLIF in traditional and embryonic mouse stem cell systems. Here we describe the expression yield of rice-derived rhLIF and the scale up production capacity. We provide further evidence of the efficacy of rice-derived rhLIF in additional stem cell systems including human neural stem cells and mouse induced pluripotent stem (iPS) cells. The expression level, biological activity, and potential for production at commercial scale of rice-derived rhLIF provides a proof-of-principal for ExpressTec-derived proteins to produce regulatory-friendly, high performance, and dependable stem cell media.

Application of recombinant human leukemia inhibitory factor (LIF) produced in rice (Oryza sativa L.) for maintenance of mouse embryonic stem cells.

Embryonic and induced pluripotent stem cells have the ability to differentiate into any somatic cell type, and thus have potential to treat a number of diseases that are currently incurable. Application of these cells for clinical or industrial uses would require an increase in production to yield adequate numbers of viable cells. However, the relatively high costs of cytokines and growth factors required for maintenance of stem cells in the undifferentiated state have the potential to limit translational research. Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, is a key regulator in the maintenance of naïve states for both human and mouse stem cells. In this study, we describe a new recombinant human LIF (rhLIF) using a plant-based (rice) expression system. We found that rice-derived rhLIF possessed the same specific activity as commercial Escherichia coli-derived LIF and was capable of supporting mouse embryonic stem cell proliferation in the undifferentiated state as evidenced from pluripotency marker level analysis. Retention of the pluripotent state was found to be indistinguishable between rice-derived rhLIF and other recombinant LIF proteins currently on the market.

Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativa L.).

BACKGROUND: Transferrin (TF) plays a critical physiological role in cellular iron delivery via the transferrin receptor (TFR)-mediated endocytosis pathway in nearly all eukaryotic organisms. Human serum TF (hTF) is extensively used as an iron-delivery vehicle in various mammalian cell cultures for production of therapeutic proteins, and is also being explored for use as a drug carrier to treat a number of diseases by employing its unique TFR-mediated endocytosis pathway. With the increasing concerns over the risk of transmission of infectious pathogenic agents of human plasma-derived TF, recombinant hTF is preferred to use for these applications. Here, we carry out comparative studies of the TFR binding, TFR-mediated endocytosis and cellular iron delivery of recombinant hTF from rice (rhTF), and evaluate its suitability for biopharmaceutical applications. RESULT: Through a TFR competition binding affinity assay with HeLa human cervic carcinoma cells (CCL-2) and Caco-2 human colon carcinoma cells (HTB-37), we show that rhTF competes similarly as hTF to bind TFR, and both the TFR binding capacity and dissociation constant of rhTF are comparable to that of hTF. The endocytosis assay confirms that rhTF behaves similarly as hTF in the slow accumulation in enterocyte-like Caco-2 cells and the rapid recycling pathway in HeLa cells. The pulse-chase assay of rhTF in Caco-2 and HeLa cells further illustrates that rice-derived rhTF possesses the similar endocytosis and intracellular processing compared to hTF. The cell culture assays show that rhTF is functionally similar to hTF in the delivery of iron to two diverse mammalian cell lines, HL-60 human promyelocytic leukemia cells (CCL-240) and murine hybridoma cells derived from a Sp2/0-Ag14 myeloma fusion partner (HB-72), for supporting their proliferation, differentiation, and physiological function of antibody production. CONCLUSION: The functional similarity between rice derived rhTF and native hTF in their cellular iron delivery, TFR binding, and TFR-mediated endocytosis and intracellular processing support that rice-derived rhTF can be used as a safe and animal-free alternative to serum hTF for bioprocessing and biopharmaceutical applications.

Biochemical and structural characterization of recombinant human serum transferrin from rice (Oryza sativa L.).

The Fe(3+) binding protein human serum transferrin (hTF) is well known for its role in cellular iron delivery via the transferrin receptor (TFR). A new application is the use of hTF as a therapy and targeted drug delivery system for a number of diseases. Recently, production of hTF in plants has been reported; such systems provide a relatively inexpensive, animal-free (eliminating potential contamination by animal pathogens) method to produce large amounts of recombinant proteins for such biopharmaceutical applications. Specifically, the production of Optiferrin (hTF produced in rice, Oryza sativa, from InVitria) has been shown to yield large amounts of functional protein for use in culture medium for cellular iron delivery to promote growth. In the present work we describe further purification (by gel filtration) and characterization of hTF produced in rice (purified Optiferrin) to determine its suitability in biopharmaceutical applications. The spectral, mass spectrometric, urea gel and kinetic analysis shows that purified Optiferrin is similar to recombinant nonglycosylated N-His tagged hTF expressed by baby hamster kidney cells and/or serum derived glycosylated hTF. Additionally, in a competitive immunoassay, iron-loaded Optiferrin is equivalent to iron-loaded N-His hTF in its ability to bind to the soluble portion of the TFR immobilized in an assay plate. As an essential requirement for any functional hTF, both lobes of purified Optiferrin bind Fe(3+) tightly yet reversibly. Although previously shown to be capable of delivering Fe(3+) to cells, the kinetics of iron release from iron-loaded Optiferrin™/sTFR and iron-loaded N-His hTF/sTFR complexes differ somewhat. We conclude that the purified Optiferrin might be suitable for consideration in biopharmaceutical applications.

Expression, purification, and characterization of recombinant human transferrin from rice (Oryza sativa L.).

Transferrin is an essential ingredient used in cell culture media due to its crucial role in regulating cellular iron uptake, transport, and utilization. It is also a promising drug carrier used to increase a drug's therapeutic index via the unique transferrin receptor-mediated endocytosis pathway. Due to the high risk of contamination with blood-borne pathogens from the use of human or animal plasma-derived transferrin, recombinant transferrin is preferred for use as a replacement for native transferrin. We expressed recombinant human transferrin in rice (Oryza sativa L.) at a high level of 1% seed dry weight (10 g/kg). The recombinant human transferrin was able to be extracted with saline buffers and then purified by a one step anion exchange chromatographic process to greater than 95% purity. The rice-derived recombinant human transferrin was shown to be not only structurally similar to the native human transferrin, but also functionally the same as native transferrin in terms of reversible iron binding and promoting cell growth and productivity. These results indicate that rice-derived recombinant human transferrin should be a safe and low cost alternative to human or animal plasma-derived transferrin for use in cell culture-based biopharmaceutical production of protein therapeutics and vaccines.

Transgenic cotton over-producing spinach sucrose phosphate synthase showed enhanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions.

Prior data indicated that enhanced availability of sucrose, a major product of photosynthesis in source leaves and the carbon source for secondary wall cellulose synthesis in fiber sinks, might improve fiber quality under abiotic stress conditions. To test this hypothesis, a family of transgenic cotton plants (Gossypium hirsutum cv. Coker 312 elite) was produced that over-expressed spinach sucrose-phosphate synthase (SPS) because of its role in regulation of sucrose synthesis in photosynthetic and heterotrophic tissues. A family of 12 independent transgenic lines was characterized in terms of foreign gene insertion, expression of spinach SPS, production of spinach SPS protein, and development of enhanced extractable V (max) SPS activity in leaf and fiber. Lines with the highest V (max) SPS activity were further characterized in terms of carbon partitioning and fiber quality compared to wild-type and transgenic null controls. Leaves of transgenic SPS over-expressing lines showed higher sucrose:starch ratio and partitioning of (14)C to sucrose in preference to starch. In two growth chamber experiments with cool nights, ambient CO(2) concentration, and limited light below the canopy, the transgenic line with the highest SPS activity in leaf and fiber had higher fiber micronaire and maturity ratio associated with greater thickness of the cellulosic secondary wall.

Analysis of ESTs from multiple Gossypium hirsutum tissues and identification of SSRs.

In an effort to expand the Gossypium hirsutum L. (cotton) expressed sequence tag (EST) database, ESTs representing a variety of tissues and treatments were sequenced. Assembly of these sequences with ESTs already in the EST database (dbEST, GenBank) identified 9675 cotton sequences not present in GenBank. Statistical analysis of a subset of these ESTs identified genes likely differentially expressed in stems, cotyledons, and drought-stressed tissues. Annotation of the differentially expressed cDNAs tentatively identified genes involved in lignin metabolism, starch biosynthesis and stress response, consistent with pathways likely to be active in the tissues under investigation. Simple sequence repeats (SSRs) were identified among these ESTs, and an inexpensive method was developed to screen genomic DNA for the presence of these SSRs. At least 69 SSRs potentially useful in mapping were identified. Selected amplified SSRs were isolated and sequenced. The sequences corresponded to the EST containing the SSRs, confirming that these SSRs will potentially map the gene represented by the EST. The ESTs containing SSRs were annotated to help identify the genes that may be mapped using these markers.

A global assembly of cotton ESTs.

Approximately 185,000 Gossypium EST sequences comprising >94,800,000 nucleotides were amassed from 30 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including drought stress and pathogen challenges. These libraries were derived from allopolyploid cotton (Gossypium hirsutum; A(T) and D(T) genomes) as well as its two diploid progenitors, Gossypium arboreum (A genome) and Gossypium raimondii (D genome). ESTs were assembled using the Program for Assembling and Viewing ESTs (PAVE), resulting in 22,030 contigs and 29,077 singletons (51,107 unigenes). Further comparisons among the singletons and contigs led to recognition of 33,665 exemplar sequences that represent a nonredundant set of putative Gossypium genes containing partial or full-length coding regions and usually one or two UTRs. The assembly, along with their UniProt BLASTX hits, GO annotation, and Pfam analysis results, are freely accessible as a public resource for cotton genomics. Because ESTs from diploid and allotetraploid Gossypium were combined in a single assembly, we were in many cases able to bioinformatically distinguish duplicated genes in allotetraploid cotton and assign them to either the A or D genome. The assembly and associated information provide a framework for future investigation of cotton functional and evolutionary genomics.

Members of a new group of chitinase-like genes are expressed preferentially in cotton cells with secondary walls.

Two homologous cotton (Gossypium hirsutum L.) genes, GhCTL1 and GhCTL2, encode members of a new group of chitinase-like proteins (called the GhCTL group) that includes other proteins from two cotton species, Arabidopsis, rice, and pea. Members of the GhCTL group are assigned to family GH19 glycoside hydrolases along with numerous authentic chitinases (http://afmb.cnrs-mrs.fr/CAZY/index.html), but the proteins have novel consensus sequences in two regions that are essential for chitinase activity and that were previously thought to be conserved. Maximum parsimony phylogenetic analyses, as well as Neighbor-Joining distance analyses, of numerous chitinases confirmed that the GhCTL group is distinct. A molecular model of GhCTL2 (based on the three-dimensional structure of a barley chitinase) had changes in the catalytic site that are likely to abolish catalytic activity while retaining potential to bind chitin oligosaccharides. RNA blot analysis showed that members of the GhCTL group had preferential expression during secondary wall deposition in cotton lint fiber. Cotton transformed with a fusion of the GhCTL2 promoter to the beta -d-glucuronidase gene showed preferential reporter gene activity in numerous cells during secondary wall deposition. Together with evidence from other researchers that mutants in an Arabidopsis gene within the GhCTL group are cellulose-deficient with phenotypes indicative of altered primary cell walls, these data suggest that members of the GhCTL group of chitinase-like proteins are essential for cellulose synthesis in primary and secondary cell walls. However, the mechanism by which they act is more likely to involve binding of chitin oligosaccharides than catalysis.

Synteny perturbations between wheat homoeologous chromosomes caused by locus duplications and deletions correlate with recombination rates.

Loci detected by Southern blot hybridization of 3,977 expressed sequence tag unigenes were mapped into 159 chromosome bins delineated by breakpoints of a series of overlapping deletions. These data were used to assess synteny levels along homoeologous chromosomes of the wheat A, B, and D genomes, in relation to both bin position on the centromere-telomere axis and the gradient of recombination rates along chromosome arms. Synteny level decreased with the distance of a chromosome region from the centromere. It also decreased with an increase in recombination rates along the average chromosome arm. There were twice as many unique loci in the B genome than in the A and D genomes, and synteny levels between the B genome chromosomes and the A and D genome homoeologues were lower than those between the A and D genome homoeologues. These differences among the wheat genomes were attributed to differences in the mating systems of wheat diploid ancestors. Synteny perturbations were characterized in 31 paralogous sets of loci with perturbed synteny. Both insertions and deletions of loci were detected and both preferentially occurred in high recombination regions of chromosomes.

Comparative DNA sequence analysis of wheat and rice genomes.

The use of DNA sequence-based comparative genomics for evolutionary studies and for transferring information from model species to crop species has revolutionized molecular genetics and crop improvement strategies. This study compared 4485 expressed sequence tags (ESTs) that were physically mapped in wheat chromosome bins, to the public rice genome sequence data from 2251 ordered BAC/PAC clones using BLAST. A rice genome view of homologous wheat genome locations based on comparative sequence analysis revealed numerous chromosomal rearrangements that will significantly complicate the use of rice as a model for cross-species transfer of information in nonconserved regions.

The organization and rate of evolution of wheat genomes are correlated with recombination rates along chromosome arms.

Genes detected by wheat expressed sequence tags (ESTs) were mapped into chromosome bins delineated by breakpoints of 159 overlapping deletions. These data were used to assess the organizational and evolutionary aspects of wheat genomes. Relative gene density and recombination rate increased with the relative distance of a bin from the centromere. Single-gene loci present once in the wheat genomes were found predominantly in the proximal, low-recombination regions, while multigene loci tended to be more frequent in distal, high-recombination regions. One-quarter of all gene motifs within wheat genomes were represented by two or more duplicated loci (paralogous sets). For 40 such sets, ancestral loci and loci derived from them by duplication were identified. Loci derived by duplication were most frequently located in distal, high-recombination chromosome regions whereas ancestral loci were most frequently located proximal to them. It is suggested that recombination has played a central role in the evolution of wheat genome structure and that gradients of recombination rates along chromosome arms promote more rapid rates of genome evolution in distal, high-recombination regions than in proximal, low-recombination regions.