Equine Polyclonal Antibodies Prevent Acute Chikungunya Virus Infection in Mice.

Chikungunya virus (CHIKV) is a mosquito-transmitted pathogen that causes chikungunya disease (CHIK); the disease is characterized by fever, muscle ache, rash, and arthralgia. This arthralgia can be debilitating and long-lasting, seriously impacting quality of life for years. Currently, there is no specific therapy available for CHIKV infection. We have developed a despeciated equine polyclonal antibody (CHIKV-EIG) treatment against CHIKV and evaluated its protective efficacy in mouse models of CHIKV infection. In immunocompromised (IFNAR-/-) mice infected with CHIKV, daily treatment for five consecutive days with CHIKV-EIG administered at 100 mg/kg starting on the day of infection prevented mortality, reduced viremia, and improved clinical condition as measured by body weight loss. These beneficial effects were seen even when treatment was delayed to 1 day after infection. In immunocompetent mice, CHIKV-EIG treatment reduced virus induced arthritis (including footpad swelling), arthralgia-associated cytokines, viremia, and tissue virus loads in a dose-dependent fashion. Collectively, these results suggest that CHIKV-EIG is effective at preventing CHIK and could be a viable candidate for further development as a treatment for human disease.

Identification and characterization of stem cell secretome-based recombinant proteins for wound healing applications.

Stem cells have been introduced as a promising therapy for acute and chronic wounds, including burn injuries. The effects of stem cell-based wound therapies are believed to result from the secreted bioactive molecules produced by stem cells. Therefore, treatments using stem cell-derived conditioned medium (CM) (referred to as secretome) have been proposed as an alternative option for wound care. However, safety and regulatory concerns exist due to the uncharacterized biochemical content and variability across different batches of CM samples. This study presents an alternative treatment strategy to mitigate these concerns by using fully characterized recombinant proteins identified by the CM analysis to promote pro-regenerative healing. This study analyzed the secretome profile generated from human placental stem cell (hPSC) cultures and identified nine predominantly expressed proteins (ANG-1, FGF-7, Follistatin, HGF, IL-6, Insulin, TGFß-1, uPAR, and VEGF) that are known to contribute to wound healing and angiogenesis. These proteins, referred to as s (CMFs), were used in combination to test the effects on human dermal fibroblasts (HDFs). Our results showed that CMF treatment increased the HDF growth and accelerated cell migration and wound closure, similar to stem cell and CM treatments. In addition, the CMF treatment promoted angiogenesis by enhancing new vessel formation. These findings suggest that the defined CMF identified by the CM proteomic analysis could be an effective therapeutic solution for wound healing applications. Our strategy eliminates the regulatory concerns present with stem cell-derived secretomes and could be developed as an off-the-shelf product for immediate wound care and accelerating healing.

Hyperimmune Targeting Staphylococcal Toxins Effectively Protect Against USA 300 MRSA Infection in Mouse Bacteremia and Pneumonia Models.

Staphylococcus aureus has been acquiring multiple drug resistance and has evolved into superbugs such as Methicillin/Vancomycin-resistant S. aureus (MRSA/VRSA) and, consequently, is a major cause of nosocomial and community infections associated with high morbidity and mortality for which no FDA-approved vaccines or biotherapeutics are available. Previous efforts targeting the surface-associated antigens have failed in clinical testing. Here, we generated hyperimmune products from sera in rabbits against six major S. aureus toxins targeted by an experimental vaccine (IBT-V02) and demonstrated significant efficacy for an anti-virulence passive immunization strategy. Extensive in vitro binding and neutralizing titers were analyzed against six extracellular toxins from individual animal sera. All IBT-V02 immunized animals elicited the maximum immune response upon the first boost dose against all pore-forming vaccine components, while for superantigen (SAgs) components of the vaccine, second and third doses of a boost were needed to reach a plateau in binding and toxin neutralizing titers. Importantly, both anti-staphylococcus hyperimmune products consisting of full-length IgG (IBT-V02-IgG) purified from the pooled sera and de-speciated F(ab')2 (IBT-V02-F(ab')2) retained the binding and neutralizing titers against IBT-V02 target toxins. F(ab')2 also exhibited cross-neutralization titers against three leukotoxins (HlgAB, HlgCB, and LukED) and four SAgs (SEC1, SED, SEK, and SEQ) which were not part of IBT-V02. F(ab')2 also neutralized toxins in bacterial culture supernatant from major clinical strains of S. aureus. In vivo efficacy data generated in bacteremia and pneumonia models using USA300 S. aureus strain demonstrated dose-dependent protection by F(ab')2. These efficacy data confirmed the staphylococcal toxins as viable targets and support the further development effort of hyperimmune products as a potential adjunctive therapy for emergency uses against life-threatening S. aureus infections.

The Ability of Zika virus Intravenous Immunoglobulin to Protect From or Enhance Zika Virus Disease.

The closely related flaviviruses, dengue and Zika, cause significant human disease throughout the world. While cross-reactive antibodies have been demonstrated to have the capacity to potentiate disease or mediate protection during flavivirus infection, the mechanisms responsible for this dichotomy are still poorly understood. To understand how the human polyclonal antibody response can protect against, and potentiate the disease in the context of dengue and Zika virus infection we used intravenous hyperimmunoglobulin (IVIG) preparations in a mouse model of the disease. Three IVIGs (ZIKV-IG, Control-Ig and Gamunex®) were evaluated for their ability to neutralize and/or enhance Zika, dengue 2 and 3 viruses in vitro. The balance between virus neutralization and enhancement provided by the in vitro neutralization data was used to predict the IVIG concentrations which could protect or enhance Zika, and dengue 2 disease in vivo. Using this approach, we were able to define the unique in vivo dynamics of complex polyclonal antibodies, allowing for both enhancement and protection from flavivirus infection. Our results provide a novel understanding of how polyclonal antibodies interact with viruses with implications for the use of polyclonal antibody therapeutics and the development and evaluation of the next generation flavivirus vaccines.

Systematic Analysis of Monoclonal Antibodies against Ebola Virus GP Defines Features that Contribute to Protection.

Antibodies are promising post-exposure therapies against emerging viruses, but which antibody features and in vitro assays best forecast protection are unclear. Our international consortium systematically evaluated antibodies against Ebola virus (EBOV) using multidisciplinary assays. For each antibody, we evaluated epitopes recognized on the viral surface glycoprotein (GP) and secreted glycoprotein (sGP), readouts of multiple neutralization assays, fraction of virions left un-neutralized, glycan structures, phagocytic and natural killer cell functions elicited, and in vivo protection in a mouse challenge model. Neutralization and induction of multiple immune effector functions (IEFs) correlated most strongly with protection. Neutralization predominantly occurred via epitopes maintained on endosomally cleaved GP, whereas maximal IEF mapped to epitopes farthest from the viral membrane. Unexpectedly, sGP cross-reactivity did not significantly influence in vivo protection. This comprehensive dataset provides a rubric to evaluate novel antibodies and vaccine responses and a roadmap for therapeutic development for EBOV and related viruses.

Novel Clostridium difficile Anti-Toxin (TcdA and TcdB) Humanized Monoclonal Antibodies Demonstrate In Vitro Neutralization across a Broad Spectrum of Clinical Strains and In Vivo Potency in a Hamster Spore Challenge Model.

Clostridium difficile (C. difficile) infection (CDI) is the main cause of nosocomial antibiotic-associated colitis and increased incidence of community-associated diarrhea in industrialized countries. At present, the primary treatment of CDI is antibiotic administration, which is effective but often associated with recurrence, especially in the elderly. Pathogenic strains produce enterotoxin, toxin A (TcdA), and cytotoxin, toxin B (TcdB), which are necessary for C. difficile induced diarrhea and gut pathological changes. Administration of anti-toxin antibodies provides an alternative approach to treat CDI, and has shown promising results in preclinical and clinical studies. In the current study, several humanized anti-TcdA and anti-TcdB monoclonal antibodies were generated and their protective potency was characterized in a hamster infection model. The humanized anti-TcdA (CANmAbA4) and anti-TcdB (CANmAbB4 and CANmAbB1) antibodies showed broad spectrum in vitro neutralization of toxins from clinical strains and neutralization in a mouse toxin challenge model. Moreover, co-administration of humanized antibodies (CANmAbA4 and CANmAbB4 cocktail) provided a high level of protection in a dose dependent manner (85% versus 57% survival at day 22 for 50 mg/kg and 20 mg/kg doses, respectively) in a hamster gastrointestinal infection (GI) model. This study describes the protective effects conferred by novel neutralizing anti-toxin monoclonal antibodies against C. difficile toxins and their potential as therapeutic agents in treating CDI.

Liquid-Liquid Phase Separation of Oil Bodies from Seeds.

Fundamentally, oil bodies are discrete storage organelles found in oilseeds, comprising a hydrophobic triacylglycerol core surrounded by a half-unit phospholipid membrane and an outer shell of specialized proteins known as oleosins. Oil bodies possess a number of attributes that were exploited by SemBioSys Genetics to isolate highly enriched fractions of oil bodies through liquid-liquid phase separation for a number of commercial applications. The current chapter provides a general guide for the isolation of oil bodies from Arabidopsis and/or safflower seed, from which protocols can be refined for different oilseed sources. For SemBioSys Genetic's recombinant technology, therapeutic proteins were covalently attached to oleosins or fused in-frame with ligands which bound oil bodies, facilitating their recovery to high levels of purity during "upstream processing" of transformed seed. Core to this technology was oil body isolation consisting of simple manipulation including homogenization of seeds to free the oil bodies, followed by the removal of insoluble fractions, and phase separation to recover the oil bodies. During oil body enrichment (an increase in oil body content concomitant with removal of impurities), a number of options and tips are provided to aid researchers in the manipulation and monitoring of these robust organelles.

Protective mAbs and Cross-Reactive mAbs Raised by Immunization with Engineered Marburg Virus GPs.

The filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. Antibodies against the filovirus surface glycoprotein (GP) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. Many monoclonal antibodies (mAbs) have been described against Ebola virus. In contrast, relatively few have been described against Marburg virus. Here we present ten mAbs elicited by immunization of mice using recombinant mucin-deleted GPs from different Marburg virus (MARV) strains. Surprisingly, two of the mAbs raised against MARV GP also cross-react with the mucin-deleted GP cores of all tested ebolaviruses (Ebola, Sudan, Bundibugyo, Reston), but these epitopes are masked differently by the mucin-like domains themselves. The most efficacious mAbs in this panel were found to recognize a novel "wing" feature on the GP2 subunit that is unique to Marburg and does not exist in Ebola. Two of these anti-wing antibodies confer 90 and 100% protection, respectively, one hour post-exposure in mice challenged with MARV.

The stripe rust resistance gene Yr10 encodes an evolutionary-conserved and unique CC-NBS-LRR sequence in wheat.

The first seedling or all-stage resistance (R) R gene against stripe rust isolated from Moro wheat (Triticum aestivum L.) using a map-based cloning approach was identified as Yr10. Clone 4B of this gene encodes a highly evolutionary-conserved and unique CC-NBS-LRR sequence. Clone 4E, a homolog of Yr10, but lacking transcription start site (TSS) and putative TATA-box and CAAT-box, is likely a non-expressed pseudogene. Clones 4B and 4E are 84% identical and divergent in the intron and the LRR domain. Gene silencing and transgenesis were used in conjunction with inoculation with differentially avirulent and virulent stripe rust strains to demonstrate Yr10 functionality. The Yr10 CC-NBS-LRR sequence is unique among known CC-NBS-LRR R genes in wheat but highly conserved homologs (E = 0.0) were identified in Aegilops tauschii and other monocots including Hordeum vulgare and Brachypodium distachyon. Related sequences were also identified in genomic databases of maize, rice, and in sorghum. This is the first report of a CC-NBS-LRR resistance gene in plants with limited homologies in its native host, but with numerous homologous R genes in related monocots that are either host or non-hosts for stripe rust. These results represent a unique example of gene evolution and dispersion across species.

Production of a Brassica napus Low-Molecular Mass Acyl-Coenzyme A-Binding Protein in Arabidopsis Alters the Acyl-Coenzyme A Pool and Acyl Composition of Oil in Seeds.

Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expression of a Brassica napus ACBP (BnACBP) complementary DNA in the developing seeds of Arabidopsis (Arabidopsis thaliana) resulted in increased levels of polyunsaturated FAs at the expense of eicosenoic acid (20:1cisΔ11) and saturated FAs in seed oil. In this study, we investigated whether alterations in the FA composition of seed oil at maturity were correlated with changes in the acyl-coenzyme A (CoA) pool in developing seeds of transgenic Arabidopsis expressing BnACBP. Our results indicated that both the acyl-CoA pool and seed oil of transgenic Arabidopsis lines expressing cytosolic BnACBP exhibited relative increases in linoleic acid (18:2cisΔ9,12; 17.9%-44.4% and 7%-13.2%, respectively) and decreases in 20:1cisΔ11 (38.7%-60.7% and 13.8%-16.3%, respectively). However, alterations in the FA composition of the acyl-CoA pool did not always correlate with those seen in the seed oil. In addition, we found that targeting of BnACBP to the endoplasmic reticulum resulted in FA compositional changes that were similar to those seen in lines expressing cytosolic BnACBP, with the most prominent exception being a relative reduction in α-linolenic acid (18:3cisΔ9,12,15) in both the acyl-CoA pool and seed oil of the former (48.4%-48.9% and 5.3%-10.4%, respectively). Overall, these data support the role of ACBP in acyl trafficking in developing seeds and validate its use as a biotechnological tool for modifying the FA composition of seed oil.

Transgenic expression of therapeutic proteins in Arabidopsis thaliana seed.

The production of therapeutic proteins in plant seed augments alternative production platforms such as microbial fermentation, cell-based systems, transgenic animals, and other recombinant plant production systems to meet increasing demands for the existing biologics, drugs under evaluation, and undiscovered therapeutics in the future. We have developed upstream purification technologies for oilseeds which are designed to cost-effectively purify therapeutic proteins amenable to conventional downstream manufacture. A very useful tool in these endeavors is the plant model system Arabidopsis thaliana. The current chapter describes the rationale and methods used to over-express potential therapeutic products in A. thaliana seed for evaluation and definitive insight into whether our production platform, Safflower, can be utilized for large-scale manufacture.

High level accumulation of gamma linolenic acid (C18:3Δ6.9,12 cis) in transgenic safflower (Carthamus tinctorius) seeds.

Gamma linolenic acid (GLA; C18:3Δ6,9,12 cis), also known as γ-Linolenic acid, is an important essential fatty acid precursor for the synthesis of very long chain polyunsaturated fatty acids and important pathways involved in human health. GLA is synthesized from linoleic acid (LA; C18:2Δ9,12 cis) by endoplasmic reticulum associated Δ6-desaturase activity. Currently sources of GLA are limited to a small number of plant species with poor agronomic properties, and therefore an economical and abundant commercial source of GLA in an existing crop is highly desirable. To this end, the seed oil of a high LA cultivated species of safflower (Carthamus tinctorius) was modified by transformation with Δ6-desaturase from Saprolegnia diclina resulting in levels exceeding 70% (v/v) of GLA. Levels around 50% (v/v) of GLA in seed oil was achieved when Δ12-/Δ6-desaturases from Mortierella alpina was over-expressed in safflower cultivars with either a high LA or high oleic (OA; C18:1Δ9 cis) background. The differences in the overall levels of GLA suggest the accumulation of the novel fatty acid was not limited by a lack of incorporation into the triacylgylcerol backbone (>66% GLA achieved), or correlated with gene dosage (GLA levels independent of gene copy number), but rather reflected the differences in Δ6-desaturase activity from the two sources. To date, these represent the highest accumulation levels of a newly introduced fatty acid in a transgenic crop. Events from these studies have been propagated and recently received FDA approval for commercialization as Sonova™400.

Expression and recovery of biologically active recombinant Apolipoprotein AI(Milano) from transgenic safflower (Carthamus tinctorius) seeds.

Apolipoprotein AI Milano (ApoAI(Milano) ) was expressed as a fusion protein in transgenic safflower seeds. High levels of expression corresponding to 7 g of ApoAI(Milano) per kilogram of seed have been identified in a line selected for commercialization. The ApoAI(Milano) fusion protein was extracted from seed using an oilbody-based process and matured in vitro prior to final purification. This yielded a Des-1,2-ApoAI(Milano) product which was confirmed by biochemical characterization including immunoreactivity against ApoAI antibodies, isoelectric point, N-terminal sequencing and electrospray mass spectrometry. Purified Des-1,2-ApoAI(Milano) readily associated with dimyristoylphosphatidylcholine in clearance assays comparable to Human ApoAI. Its biological activity was assessed by cholesterol efflux assays using Des-1,2-ApoAI(Milano) :1-palmitoyl-2-oleoyl phosphatidylcholine complexes in vitro and in vivo. This study has established that high levels of biologically functional ApoAI(Milano) can be produced using a plant-based expression system.

Seed-based expression systems for plant molecular farming.

The evolution of the seed system provides enormous adaptability to the gymnosperms and angiosperms, because of the properties of dormancy, nutrient storage and seedling vigour. Many of the unique properties of seeds can be exploited in molecular farming applications, particularly where it is desirable to produce large quantities of a recombinant protein. Seeds of transgenic plants have been widely used to generate a raw material for the extraction and isolation of proteins and polypeptides, which can be processed into valuable biopharmaceuticals. The factors that control high-level accumulation of recombinant proteins in seed are reviewed in the following paragraphs. These include promoters and enhancers, which regulate transcript abundance. However, it is shown that subcellular trafficking and targeting of the desired polypeptides or proteins play a crucial role in their accumulation at economically useful levels. Seeds have proven to be versatile hosts for recombinant proteins of all types, including peptides or short and long polypeptides as well as complex, noncontiguous proteins like antibodies and other immunoglobulins. The extraction and recovery of recombinant proteins from seeds is greatly assisted by their dormancy properties, because this allows for long-term stability of stored products including recombinant proteins and a decoupling of processing from the growth and harvest cycles. Furthermore, the low water content and relatively low bioload of seeds help greatly in designing cost-effective manufacturing processes for the desired active pharmaceutical ingredient. The development of cGMP processes based on seed-derived materials has only been attempted by a few groups to date, but we provide a review of the key issues and criteria based on interactions with Food and Drug Administration and European Medicines Agency. This article uses 'case studies' to highlight the utility of seeds as vehicles for pharmaceutical production including: insulin, human growth hormone, lysozyme and lactoferrin. These examples serve to illustrate the preclinical and, in one case, clinical information required to move these plant-derived molecules through the research phase and into the regulatory pathway en route to eventual approval.

A 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus enhances acyl exchange between acyl-CoA and phosphatidylcholine.

The gene encoding a 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus was over-expressed in developing seeds of Arabidopsis thaliana. Biochemical analysis of T(2) and T(3) A. thaliana seeds revealed a significant increase in polyunsaturated fatty acids (FAs) (18:2(cisDelta9,12) and 18:3(cisDelta9,12,15)) at the expense of very long monounsaturated FA (20:1(cisDelta11)) and saturated FAs. In vitro assays demonstrated that recombinant B. napus ACBP (rBnACBP) strongly increases the formation of phosphatidylcholine (PC) in the absence of added lysophosphatidylcholine in microsomes from DeltaYOR175c yeast expressing A. thaliana lysophosphatidylcholine acyltransferase (AthLPCAT) cDNA or in microsomes from microspore-derived cell suspension cultures of B. napus L. cv. Jet Neuf. rBnACBP or bovine serum albumin (BSA) were also shown to be crucial for AthLPCAT to catalyse the transfer of acyl group from PC into acyl-CoA in vitro. These data suggest that the cytosolic 10-kDa ACBP has an effect on the equilibrium between metabolically active acyl pools (acyl-CoA and phospholipid pools) involved in FA modifications and triacylglycerol bioassembly in plants. Over-expression of ACBP during seed development may represent a useful biotechnological approach for altering the FA composition of seed oil.

Acyl-CoA-binding and self-associating properties of a recombinant 13.3 kDa N-terminal fragment of diacylglycerol acyltransferase-1 from oilseed rape.

BACKGROUND: Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to generate triacylglycerol and CoA. The deduced amino acid sequence of cDNAs encoding DGAT1 from plants and mammals exhibit a hydrophilic N-terminal region followed by a number of potential membrane-spanning segments, which is consistent with the membrane-bound nature of this enzyme family. In order to gain insight into the structure/function properties of DGAT1 from Brassica napus (BnDGAT1), we produced and partially characterized a recombinant polyHis-tagged N-terminal fragment of the enzyme, BnDGAT1(1-116)His6, with calculated molecular mass of 13,278 Da. RESULTS: BnDGAT1(1-116)His6 was highly purified from bacterial lysate and plate-like monoclinic crystals were grown using this preparation. Lipidex-1000 binding assays and gel electrophoresis indicated that BnDGAT1(1-116)His6 interacts with long chain acyl-CoA. The enzyme fragment displayed enhanced affinity for erucoyl (22:1cisDelta13)-CoA over oleoyl (18:1cisDelta9)-CoA, and the binding process displayed positive cooperativity. Gel filtration chromatography and cross-linking studies indicated that BnDGAT1(1-116)His6 self-associated to form a tetramer. Polyclonal antibodies raised against a peptide of 15 amino acid residues representing a segment of BnDGAT1(1-116)His6 failed to react with protein in microsomal vesicles following treatment with proteinase K, suggesting that the N-terminal fragment of BnDGAT1 was localized to the cytosolic side of the ER. CONCLUSION: Collectively, these results suggest that BnDGAT1 may be allosterically modulated by acyl-CoA through the N-terminal region and that the enzyme self-associates via interactions on the cytosolic side of the ER.

Transgenic expression and recovery of biologically active recombinant human insulin from Arabidopsis thaliana seeds.

The increased incidence of diabetes, coupled with the introduction of alternative delivery methods that rely on higher doses, is expected to result in a substantial escalation in the demand for affordable insulin in the future. Limitations in the capacity and economics of production will make it difficult for current manufacturing technologies to meet this demand. We have developed a novel expression and recovery technology for the economical manufacture of biopharmaceuticals from oilseeds. Using this technology, recombinant human precursor insulin was expressed in transgenic plants. Plant-derived insulin accumulates to significant levels in transgenic seed (0.13% total seed protein) and can be enzymatically treated in vitro to generate a product with a mass identical to that of the predicted product, DesB(30)-insulin. The biological activity of this product in vivo and in vitro was demonstrated using an insulin tolerance test in mice and phosphorylation assay performed in a mammalian cell culture system, respectively.

The accumulation of oleosins determines the size of seed oilbodies in Arabidopsis.

We investigated the role of the oilbody proteins in developing and germinating Arabidopsis thaliana seeds. Seed oilbodies are simple organelles comprising a matrix of triacylglycerol surrounded by a phospholipid monolayer embedded and covered with unique proteins called oleosins. Indirect observations have suggested that oleosins maintain oilbodies as small single units preventing their coalescence during seed desiccation. To understand the role of oleosins during seed development or germination, we created lines of Arabidopsis in which a major oleosin is ablated or severely attenuated. This was achieved using RNA interference techniques and through the use of a T-DNA insertional event, which appears to interrupt the major (18 kD) seed oleosin gene of Arabidopsis and results in ablation of expression. Oleosin suppression resulted in an aberrant phenotype of embryo cells that contain unusually large oilbodies that are not normally observed in seeds. Changes in the size of oilbodies caused disruption of storage organelles, altering accumulation of lipids and proteins and causing delay in germination. The aberrant phenotypes were reversed by reintroducing a recombinant oleosin. Based on this direct evidence, we have shown that oleosins are important proteins in seed tissue for controlling oilbody structure and lipid accumulation.

Characterization of cDNAs encoding diacylglycerol acyltransferase from cultures of Brassica napus and sucrose-mediated induction of enzyme biosynthesis.

cDNAs encoding acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20), designated BnDGAT1 and BnDGAT2, were obtained from a microspore-derived cell suspension culture of oilseed rape (Brassica napus L. cv Jet Neuf). BnDGAT2 shares a very high level of identity with BnDGAT1, but is a smaller protein lacking the relatively hydrophilic N-terminal segment found in BnDGAT1. Both transcripts were produced in the cell suspension cultures and the cDNAs were functionally expressed in transformed yeast (Pichia pastoris) cells. Sucrose-mediated changes in triacylglycerol (TAG) metabolism and expression of BnDGAT1 were examined in the cell suspension cultures following transfer of cells from media containing 6% (w/v) sucrose to media containing 14% sucrose. TAG content and DGAT activity of the cells increased transiently within the first 12 h after transfer (HAT). The rapid decline in TAG content observed at 12 HAT was inversely related to an increase in TAG lipase (EC 3.1.1.3) activity. The transient increases in TAG content and DGAT activity correlated with the elevated amounts of BnDGAT1 polypeptide. Transcript levels were also induced, but levels of mRNA encoding BnDGAT1 were not tightly correlated with DGAT activity and amount of polypeptide suggesting some control of expression at the post-transcriptional level. In general, the rapid changes in TAG content were closely associated with the changes in the activity of TAG-metabolizing enzymes and expression of BnDGAT1.