Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris

Yeast is capable of performing posttranslational modifications, such as N- or O-glycosylation. It has been demonstrated that N-glycans play critical biological roles in therapeutic glycoproteins by modulating pharmacokinetics and pharmacodynamics. However, N-glycan sites on recombinant glycoproteins produced in yeast can be underglycosylated, and hence, not completely occupied. Genomic homology analysis indicates that the Pichia pastoris oligosaccharyltransferase (OST) complex consists of multiple subunits, including OST1, OST2, OST3, OST4, OST5, OST6, STT3, SWP1, and WBP1. Monoclonal antibodies produced in P. pastoris show that N-glycan site occupancy ranges from 75–85 % and is affected mainly by the OST function, and in part, by process conditions. In this study, we demonstrate that N-glycan site occupancy of antibodies can be improved to greater than 99 %, comparable to that of antibodies produced in mammalian cells (CHO), by overexpressing Leishmania major STT3D (LmSTT3D) under the control of an inducible alcohol oxidase 1 (AOX1) promoter. N-glycan site occupancy of non-antibody glycoproteins such as recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was also significantly improved, suggesting that LmSTT3D has broad substrate specificity. These results suggest that the glycosylation status of recombinant proteins can be improved by heterologous STT3 expression, which will allow for the customization of therapeutic protein profiles.     

Automated measurement of site‐specific N‐glycosylation occupancy with SWATH‐MS

Asparagine‐linked glycosylation is a common post‐translational modification of proteins catalyzed by oligosaccharyltransferase that is important in regulating many aspects of protein function. Analysis of protein glycosylation, including glycoproteomic measurement of the site‐specific extent of glycosylation, remains challenging. Here, we developed methods combining enzymatic deglycosylation and protease digestion with SWATH‐MS to enable automated measurement of site‐specific occupancy at many glycosylation sites. Deglycosylation with peptide‐endoglycosidase H, leaving a remnant N‐acetylglucosamine on asparagines previously carrying high‐mannose glycans, followed by trypsin digestion allowed robust automated measurement of occupancy at many sites. Combining deglycosylation with the more general peptide‐N‐glycosidase F enzyme with AspN protease digest allowed robust automated differentiation of nonglycosylated and deglycosylated forms of a given glycosylation site. Ratiometric analysis of deglycosylated peptides and the total intensities of all peptides from the corresponding proteins allowed relative quantification of site‐specific glycosylation occupancy between yeast strains with various isoforms of oligosaccharyltransferase. This approach also allowed robust measurement of glycosylation sites in human salivary glycoproteins. This method for automated relative quantification of site‐specific glycosylation occupancy will be a useful tool for research with model systems and clinical samples.     

Identification of cell culture conditions to control N‐glycosylation site‐occupancy of recombinant glycoproteins expressed in CHO cells

The effect of different cell culture conditions on N‐glycosylation site‐occupancy has been elucidated for two different recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells, recombinant human tissue plasminogen activator (t‐PA) and a recombinant enzyme (glycoprotein 2—GP2). Both molecules contain a N‐glycosylation site that is variably occupied. Different environmental factors that affect the site‐occupancy (the degree of occupied sites) of these molecules were identified. Supplementing the culture medium with additional manganese or iron increased the fraction of fully occupied t‐PA (type I t‐PA) by approximately 2.5–4%. Decreasing the cultivation temperature from 37 to 33°C or 31°C gradually increased site‐occupancy of t‐PA up to 4%. The addition of a specific productivity enhancer, butyrate, further increased site‐occupancy by an additional 1% under each cultivation temperature tested. In addition, the thyroid hormones triiodothyronine and thyroxine increased site‐occupancy of t‐PA compared to control conditions by about 2%. In contrast, the addition of relevant nucleoside precursor molecules involved in N‐glycan biosynthesis (e.g., uridine, guanosine, mannose) either had no effect or slightly reduced site‐occupancy. For the recombinant enzyme (GP2), it was discovered that culture pH and the timing of butyrate addition can be used to control N‐glycan site‐occupancy within a specific range. An increase in culture pH correlated with a decrease in site‐occupancy. Similarly, delaying the timing for butyrate addition also decreased site‐occupancy of this molecule. These results highlight the importance of understanding how cell culture conditions and media components can affect the product quality of recombinant glycoproteins expressed in mammalian cell cultures. Furthermore, the identification of relevant factors will enable one to control product quality attributes, specifically N‐glycan site‐occupancy, within a specific range when applied appropriately. Biotechnol. Bioeng. 2009;103: 1164–1175. © 2009 Wiley Periodicals, Inc.     

Arabidopsis thaliana FLA4 functions as a glycan‐stabilized soluble factor via its carboxy‐proximal Fasciclin 1 domain

Fasciclin‐like arabinogalactan proteins (FLAs) are involved in numerous important functions in plants but the relevance of their complex structure to physiological function and cellular fate is unresolved. Using a fully functional fluorescent version of Arabidopsis thaliana FLA4 we show that this protein is localized at the plasma membrane as well as in endosomes and soluble in the apoplast. FLA4 is likely to be GPI‐anchored, is highly N‐glycosylated and carries two O‐glycan epitopes previously associated with arabinogalactan proteins. The activity of FLA4 was resistant against deletion of the amino‐proximal fasciclin 1 domain and was unaffected by removal of the GPI‐modification signal, a highly conserved N‐glycan or the deletion of predicted O‐glycosylation sites. Nonetheless these structural changes dramatically decreased endoplasmic reticulum (ER)‐exit and plasma membrane localization of FLA4, with N‐glycosylation acting at the level of ER‐exit and O‐glycosylation influencing post‐secretory fate. We show that FLA4 acts predominantly by molecular interactions involving its carboxy‐proximal fasciclin 1 domain and that its amino‐proximal fasciclin 1 domain is required for stabilization of plasma membrane localization. FLA4 functions as a soluble glycoprotein via its carboxy‐proximal Fas1 domain and its normal cellular trafficking depends on N‐ and O‐glycosylation.     

N‐glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

Studies of protein N‐glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N‐glycan maturation in the glycosylation machinery. The inactivation of the individual genes resulted in altered N‐glycan patterns as documented using mass spectrometry and glycan‐recognising antibodies, indicating successful identification of null mutations in the target glyco‐genes. For example, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3‐fucosyltransferase (Lj3fuct) mutant completely lacked α1,3‐core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N‐glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N‐acetylglucosaminyltransferase I, and α1,3‐fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N‐glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N‐glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian‐like N‐glycosylation features.     

Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases

Asparagine‐linked glycosylation is catalysed by oligosaccharyltransferase (OTase). In Trypanosoma brucei OTase activity is catalysed by single‐subunit enzymes encoded by three paralogous genes of which TbSTT3B and TbSTT3C can complement a yeast Δstt3 mutant. The two enzymes have overlapping but distinct peptide acceptor specificities, with TbSTT3C displaying an enhanced ability to glycosylate sites flanked by acidic residues. TbSTT3A and TbSTT3B, but not TbSTT3C, are transcribed in the bloodstream and procyclic life cycle stages of T. brucei. Selective knockdown and analysis of parasite protein N‐glycosylation showed that TbSTT3A selectively transfers biantennary Man₅GlcNAc₂ to specific glycosylation sites whereas TbSTT3B selectively transfers triantennary Man₉GlcNAc₂ to others. Analysis of T. brucei glycosylation site occupancy showed that TbSTT3A and TbSTT3B glycosylate sites in acidic to neutral and neutral to basic regions of polypeptide, respectively. This embodiment of distinct specificities in single‐subunit OTases may have implications for recombinant glycoprotein engineering. TbSTT3A and TbSTT3B could be knocked down individually, but not collectively, in tissue culture. However, both were independently essential for parasite growth in mice, suggesting that inhibiting protein N‐glycosylation could have therapeutic potential against trypanosomiasis.     

The production of human glucocerebrosidase in glyco‐engineered Nicotiana benthamiana plants

For the production of therapeutic proteins in plants, the presence of β1,2‐xylose and core α1,3‐fucose on plants’ N‐glycan structures has been debated for their antigenic activity. In this study, RNA interference (RNAi) technology was used to down‐regulate the endogenous N‐acetylglucosaminyltransferase I (GNTI) expression in Nicotiana benthamiana. One glyco‐engineered line (NbGNTI‐RNAi) showed a strong reduction of plant‐specific N‐glycans, with the result that as much as 90.9% of the total N‐glycans were of high‐mannose type. Therefore, this NbGNTI‐RNAi would be a promising system for the production of therapeutic glycoproteins in plants. The NbGNTI‐RNAi plant was cross‐pollinated with transgenic N. benthamiana expressing human glucocerebrosidase (GC). The recombinant GC, which has been used for enzyme replacement therapy in patients with Gaucher's disease, requires terminal mannose for its therapeutic efficacy. The N‐glycan structures that were presented on all of the four occupied N‐glycosylation sites of recombinant GC in NbGNTI‐RNAi plants (GC^gnt1) showed that the majority (ranging from 73.3% up to 85.5%) of the N‐glycans had mannose‐type structures lacking potential immunogenic β1,2‐xylose and α1,3‐fucose epitopes. Moreover, GC^gnt1 could be taken up into the macrophage cells via mannose receptors, and distributed and taken up into the liver and spleen, the target organs in the treatment of Gaucher's disease. Notably, the NbGNTI‐RNAi line, producing GC, was stable and the NbGNTI‐RNAi plants were viable and did not show any obvious phenotype. Therefore, it would provide a robust tool for the production of GC with customized N‐glycan structures.     

Analysis of glycosylation site occupancy reveals a role for Ost3p and Ost6p in site-specific N-glycosylation efficiency

Asparagine-linked glycosylation is the most common post-translational modification of proteins catalyzed in eukaryotes by the multiprotein complex oligosaccharyltransferase. Apart from the catalytic Stt3p, the roles of the subunits are ill defined. Here we describe functional investigations of the Ost3/6p components of the yeast enzyme. We developed novel analytical tools to quantify glycosylation site occupancy by enriching glycoproteins bound to the yeast polysaccharide cell wall, tagging glycosylated asparagines using endoglycosidase H glycan release, and detecting peptides and glycopeptides with LC-ESI-MS/MS. We found that the paralogues Ost3p and Ost6p were required for efficient glycosylation of distinct defined glycosylation sites. Our results describe a novel method for relative quantification of glycosylation occupancy in the genetically tractable yeast system and show that eukaryotic oligosaccharyltransferase isoforms have different activities toward protein substrates at the level of individual glycosylation sites.     

Production of monoclonal antibodies with a controlled N‐glycosylation pattern in seeds of Arabidopsis thaliana

Seed‐specific expression is an appealing alternative technology for the production of recombinant proteins in transgenic plants. Whereas attractive yields of recombinant proteins have been achieved by this method, little attention has been paid to the intracellular deposition and the quality of such products. Here, we demonstrate a comparative study of two antiviral monoclonal antibodies (mAbs) (HA78 against Hepatitis A virus; 2G12 against HIV) expressed in seeds of Arabidopsis wild‐type (wt) plants and glycosylation mutants lacking plant specific N‐glycan residues. We demonstrate that 2G12 is produced with complex N‐glycans at great uniformity in the wt as well as in the glycosylation mutant, carrying a single dominant glycosylation species, GnGnXF and GnGn, respectively. HA78 in contrast, contains additionally to complex N‐glycans significant amounts of oligo‐mannosidic structures, which are typical for endoplasmic reticulum (ER)‐retained proteins. A detailed subcellular localization study demonstrated the deposition of both antibodies virtually exclusively in the extracellular space, illustrating their efficient secretion. In addition, although a KDEL‐tagged version of 2G12 exhibited an ER‐typical N‐glycosylation pattern, it was surprisingly detected in protein storage vacuoles. The different antibody variants showed different levels of degradation with hardly any degradation products detectable for HA78 carrying GnGnXF glycans. Finally, we demonstrate functional integrity of the HA78 and 2G12 glycoforms using viral inhibition assays. Our data therefore demonstrate the usability of transgenic seeds for the generation of mAbs with a controlled N‐glycosylation pattern, thus expanding the possibilities for the production of optimally glycosylated proteins with enhanced biological activities for the use as human therapeutics.     

BGAL1 depletion boosts the level of β‐galactosylation of N‐ and O‐glycans in N. benthamiana

Glyco‐design of proteins is a powerful tool in fundamental studies of structure–function relationship and in obtaining profiles optimized for efficacy of therapeutic glycoproteins. Plants, particularly Nicotiana benthamiana, are attractive hosts to produce recombinant glycoproteins, and recent advances in glyco‐engineering facilitate customized N‐glycosylation of plant‐derived glycoproteins. However, with exception of monoclonal antibodies, homogenous human‐like β1,4‐galactosylation is very hard to achieve in recombinant glycoproteins. Despite significant efforts to optimize the expression of β1,4‐galactosyltransferase, many plant‐derived glycoproteins still exhibit incomplete processed N‐glycans with heterogeneous terminal galactosylation. The most obvious suspects to be involved in trimming terminal galactose residues are β‐galactosidases (BGALs) from the glycosyl hydrolase family GH35. To elucidate the so far uncharacterized mechanisms leading to the trimming of terminal galactose residues from glycans of secreted proteins, we studied a N. benthamiana BGAL known to be active in the apoplast (NbBGAL1). Here, we determined the NbBGAL1 subcellular localization, substrate specificity and in planta biological activity. We show that NbBGAL1 can remove β1,4‐ and β1,3‐galactose residues on both N‐ and O‐glycans. Transient BGAL1 down‐regulation by RNA interference (RNAi) and BGAL1 depletion by genome editing drastically reduce β‐galactosidase activity in N. benthamiana and increase the amounts of fully galactosylated complex N‐glycans on several plant‐produced glycoproteins. Altogether, our data demonstrate that NbBGAL1 acts on galactosylated complex N‐glycans of plant‐produced glycoproteins.     

The N‐glycan on Asn54 affects the atypical N‐glycan composition of plant‐produced interleukin‐22, but does not influence its activity

Human interleukin‐22 (IL‐22) is a member of the IL‐10 cytokine family that has recently been shown to have major therapeutic potential. IL‐22 is an unusual cytokine as it does not act directly on immune cells. Instead, IL‐22 controls the differentiation, proliferation and antimicrobial protein expression of epithelial cells, thereby maintaining epithelial barrier function. In this study, we transiently expressed human IL‐22 in Nicotiana benthamiana plants and investigated the role of N‐glycosylation on protein folding and biological activity. Expression levels of IL‐22 were up to 5.4 μg/mg TSP, and N‐glycan analysis revealed the presence of the atypical Lewis A structure. Surprisingly, upon engineering of human‐like N‐glycans on IL‐22 by co‐expressing mouse FUT8 in ΔXT/FT plants a strong reduction in Lewis A was observed. Also, core α1,6‐fucoylation did not improve the biological activity of IL‐22. The combination of site‐directed mutagenesis of Asn54 and in vivo deglycosylation with PNGase F also revealed that N‐glycosylation at this position is not required for proper protein folding. However, we do show that the presence of a N‐glycan on Asn54 contributes to the atypical N‐glycan composition of plant‐produced IL‐22 and influences the N‐glycan composition of N‐glycans on other positions. Altogether, our data demonstrate that plants offer an excellent tool to investigate the role of N‐glycosylation on folding and activity of recombinant glycoproteins, such as IL‐22.     

All in one: Leishmania major STT3 proteins substitute for the whole oligosaccharyltransferase complex in Saccharomyces cerevisiae

The transfer of lipid-linked oligosaccharide to asparagine residues of polypeptide chains is catalyzed by oligosaccharyltransferase (OTase). In most eukaryotes, OTase is a hetero-oligomeric complex composed of eight different proteins, in which the STT3 component is believed to be the catalytic subunit. In the parasitic protozoa Leishmania major, four STT3 paralogues, but no homologues to the other OTase components seem to be encoded in the genome. We expressed each of the four L. major STT3 proteins individually in Saccharomyces cerevisiae and found that three of them, LmSTT3A, LmSTT3B, and LmSTT3D, were able to complement a deletion of the yeast STT3 locus. Furthermore, LmSTT3D expression suppressed the lethal phenotype of single and double deletions in genes encoding other essential OTase subunits. LmSTT3 proteins did not incorporate into the yeast OTase complex but formed a homodimeric enzyme, capable of replacing the endogenous, multimeric enzyme of the yeast cell. Therefore, these protozoan OTases resemble the prokaryotic enzymes with respect to their architecture, but they used substrates typical for eukaryotic cells: N-X-S/T sequons in proteins and dolicholpyrophosphate-linked high mannose oligosaccharides.     

Brassica rapa hairy root based expression system leads to the production of highly homogenous and reproducible profiles of recombinant human alpha‐L‐iduronidase

The Brassica rapa hairy root based expression platform, a turnip hairy root based expression system, is able to produce human complex glycoproteins such as the alpha—L—iduronidase (IDUA) with an activity similar to the one produced by Chinese Hamster Ovary (CHO) cells. In this article, a particular attention has been paid to the N‐ and O‐glycosylation that characterize the alpha‐L‐iduronidase produced using this hairy root based system. This analysis showed that the recombinant protein is characterized by highly homogeneous post translational profiles enabling a strong batch to batch reproducibility. Indeed, on each of the 6 N‐glycosylation sites of the IDUA, a single N‐glycan composed of a core Man₃GlcNAc₂ carrying one beta(1,2)‐xylose and one alpha(1,3)‐fucose epitope (M3XFGN2) was identified, highlighting the high homogeneity of the production system. Hydroxylation of proline residues and arabinosylation were identified during O‐glycosylation analysis, still with a remarkable reproducibility. This platform is thus positioned as an effective and consistent expression system for the production of human complex therapeutic proteins.     

Control of the pattern‐recognition receptor EFR by an ER protein complex in plant immunity

In plant innate immunity, the surface‐exposed leucine‐rich repeat receptor kinases EFR and FLS2 mediate recognition of the bacterial pathogen‐associated molecular patterns EF‐Tu and flagellin, respectively. We identified the Arabidopsis stromal‐derived factor‐2 (SDF2) as being required for EFR function, and to a lesser extent FLS2 function. SDF2 resides in an endoplasmic reticulum (ER) protein complex with the Hsp40 ERdj3B and the Hsp70 BiP, which are components of the ER‐quality control (ER‐QC). Loss of SDF2 results in ER retention and degradation of EFR. The differential requirement for ER‐QC components by EFR and FLS2 could be linked to N‐glycosylation mediated by STT3a, a catalytic subunit of the oligosaccharyltransferase complex involved in co‐translational N‐glycosylation. Our results show that the plasma membrane EFR requires the ER complex SDF2–ERdj3B–BiP for its proper accumulation, and provide a demonstration of a physiological requirement for ER‐QC in transmembrane receptor function in plants. They also provide an unexpected differential requirement for ER‐QC and N‐glycosylation components by two closely related receptors.     

N‐glycosylation proteomic characterization and cross‐species comparison of milk fat globule membrane proteins from mammals

Glycosylation of proteins has been implicated in various biological functions and has received much attention; however, glycoprotein components and inter‐species complexity have not yet been elucidated fully in milk proteins. N‐linked glycosylation sites and glycoproteins in milk fat globule membrane (MFGM) fractions were investigated by combining N‐glycosylated peptides enrichment and high‐accuracy Q Exactive identification, to map the N‐glycoproteome profiles in Holstein and Jersey cows, buffaloes, yaks, goats, camels, horses, and humans. A total of 399 N‐glycoproteins with 677 glycosylation sites were identified in the MFGM fractions of the studied mammals. Most glycosylation sites in humans were classified as known and those in the other studied mammals as unknown, according to Swiss‐Prot annotations. Functionally, most of the identified glycoproteins were associated with the ‘response to stimulus’ GO category. N‐glycosylated protein components of MFGM fractions from Holstein and Jersey cows, buffaloes, yaks, and goats were more similar to each other compared with those of camels, horses and human. The findings increased the number of known N‐glycosylation sites in the milk from dairy animal species, revealed the complexity of the MFGM glycoproteome, and provided useful information to further explore the mechanism of MFGM glycoproteins biosynthesis among the studied mammals.     

Improving the baculovirus expression vector system with vankyrin‐enhanced technology

The baculovirus expression vector system (BEVS) is a widely used platform for the production of recombinant eukaryotic proteins. However, the BEVS has limitations in comparison to other higher eukaryotic expression systems. First, the insect cell lines used in the BEVS cannot produce glycoproteins with complex‐type N‐glycosylation patterns. Second, protein production is limited as cells die and lyse in response to baculovirus infection. To delay cell death and lysis, we transformed several insect cell lines with an expression plasmid harboring a vankyrin gene (P‐vank‐1), which encodes an anti‐apoptotic protein. Specifically, we transformed Sf9 cells, Trichoplusia ni High Five^TM cells, and SfSWT‐4 cells, which can produce glycoproteins with complex‐type N‐glycosylation patterns. The latter was included with the aim to increase production of glycoproteins with complex N‐glycans, thereby overcoming the two aforementioned limitations of the BEVS. To further increase vankyrin expression levels and further delay cell death, we also modified baculovirus vectors with the P‐vank‐1 gene. We found that cell lysis was delayed and recombinant glycoprotein yield increased when SfSWT‐4 cells were infected with a vankyrin‐encoding baculovirus. A synergistic effect in elevated levels of recombinant protein production was observed when vankyrin‐expressing cells were combined with a vankyrin‐encoding baculovirus. These effects were observed with various model proteins including medically relevant therapeutic proteins. In summary, we found that cell lysis could be delayed and recombinant protein yields could be increased by using cell lines constitutively expressing vankyrin or vankyrin‐encoding baculovirus vectors. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1496–1507, 2017     

A two‐component enzyme complex is required for dolichol biosynthesis in tomato

Dolichol plays an indispensable role in the N‐glycosylation of eukaryotic proteins. As proteins enter the secretory pathway they are decorated by a ‘glycan’, which is preassembled onto a membrane‐anchored dolichol molecule embedded within the endoplasmic reticulum (ER). Genetic and biochemical evidence in yeast and animals indicate that a cis‐prenyltransferase (CPT) is required for dolichol synthesis, but also point to other factor(s) that could be involved. In this study, RNAi‐mediated suppression of one member of the tomato CPT family (SlCPT3) resulted in a ~60% decrease in dolichol content. We further show that the involvement of SlCPT3 in dolichol biosynthesis requires the participation of a distantly related partner protein, designated as CPT‐binding protein (SlCPTBP), which is a close homolog of the human Nogo‐B receptor. Yeast two‐hybrid and co‐immunoprecipitation assays demonstrate that SlCPT3 and its partner protein interact in vivo and that both SlCPT3 and SlCPTBP are required to complement the growth defects and dolichol deficiency of the yeast dolichol mutant, rer2?. Co‐expression of SlCPT3 and SlCPTBP in yeast and in E. coli confirmed that dolichol synthase activity strictly requires both proteins. Finally, organelle isolation and in vivo localization of fluorescent protein fusions showed that both SlCPT3 and SlCPTBP localize to the ER, the site of dolichol accumulation and synthesis in eukaryotes.     

Mouse fat storage‐inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants

Fat storage‐inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)‐localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. Here, we tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension‐cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids in leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. When expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER‐LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ER‐vesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. These results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.