Catherine Navarre

Production of antibodies in plants: status after twenty years.

Thanks to their potential to bind virtually all types of molecules; monoclonal antibodies are in increasing demand as therapeutics and diagnostics. To overcome the overloading of current production facilities, alternative expression systems have been developed, of which plants appear the most promising. In this review, we focus on the expression of monoclonal IgG or IgM in plant species. We analyse the data for 32 different antibodies expressed in various ways, differing in DNA construction, transformation method, signal peptide source, presence or absence of an endoplasmic reticulum retention sequence, host species and the organs tested, together resulting in 98 reported combinations. A large heterogeneity is found in the quantity and quality of the antibody produced. We discuss in more detail the strategy used to express both chains, the nature of the transcription promoters, subcellular localization and unintended proteolysis, when encountered.

Membrane hyperpolarization and salt sensitivity induced by deletion of PMP3, a highly conserved small protein of yeast plasma membrane

Yeast plasma membranes contain a small 55 amino acid hydrophobic polypeptide, Pmp3p, which has high sequence similarity to a novel family of plant polypeptides that are overexpressed under high salt concentration or low temperature treatment. The PMP3 gene is not essential under normal growth conditions. However, its deletion increases the plasma membrane potential and confers sensitivity to cytotoxic cations, such as Na+ and hygromycin B. Interestingly, the disruption of PMP3 exacerbates the NaCl sensitivity phenotype of a mutant strain lacking the Pmr2p/Enap Na+‐ATPases and the Nha1p Na+/H+ antiporter, and suppresses the potassium dependency of a strain lacking the K+ transporters, Trk1p and Trk2p. All these phenotypes could be reversed by the addition of high Ca2+ concentration to the medium. These genetic interactions indicate that the major effect of the PMP3 deletion is a hyperpolarization of the plasma membrane potential that probably promotes a non‐specific influx of monovalent cations. Expression of plant RCI2A in yeast could substitute for the loss of Pmp3p, indicating a common role for Pmp3p and the plant homologue.

Gene Inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 Suspension Cells

Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices. Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells.

Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco

Rhizosecretion is an attractive technology for the production of recombinant proteins from transgenic plants. However, to date, yields of plantderived recombinant pharmaceuticals by this method have been too low for commercial viability. Studies conducted focused on three transgenic plant lines grown in hydroponic culture medium, two expressing monoclonal antibodies Guys 13 and 4E10 and one expressing a small microbicide polypeptide cyanovirin‐N. Rhizosecretion rates increased significantly by the addition of the plant growth regulator α‐naphthalene acetic acid. The maximum rhizosecretion rates achieved were 58 Hg/g root dry weight/24 h for Guys 13, 10.43 μg/g root dry weight/24 h for 4E10, and 766 μg/g root dry weight/24 h for cyanovirin‐N, the highest figures so far reported for a full‐length antibody and a recombinant protein, respectively. The plant growth regulators indolebutyric acid, 6–benzylaminopurine, and kinetin were also demonstrated to increase rhizosecretion of Guys 13. The effect of the growth regulators differed, as α‐naphthalene acetic acid and indole‐butyric acid increased the root dry weight of hydroponic plants, whereas the cytokinins benzylaminopurine and kinetin increased rhizosecretion without affecting root mass. A comparative glycosylation analysis between MAb Guys 13 purified from either hydroponic culture medium or from leaf extracts demonstrated a similar pattern of glycosylation comprising high mannose to complex glycoforms. Analysis of the hydroponic culture medium at harvest revealed significantly lower and less complex levels of proteolytic enzymes, in comparison with leaf extracts, which translated to a higher proportion of intact Guys 13 IgG in relation to other IgG products. Hydroponic medium could be added directly to a chromatography column for affinity purification, allowing simple and rapid production of high purity Guys 13 antibody. In addition to the attractiveness of controlled cultivation within a contained environment for pharmaceutical‐producing plants, this study demonstrates advantages with respect to the quality and downstream purification of recombinant proteins.—Drake, P. M. W., Barbi, T., Sexton, A., McGowan, E., Stadlmann, J., Navarre, C., Paul, M. J., Ma, J. K.‐C. Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco. FASEB J. 23, 3581–3589 (2009). www.fasebj.org

Subproteomics: identification of plasma membrane proteins from the yeast Saccharomyces cerevisiae.

As a consequence of their poor solubility during isoelectric focusing, integral membrane proteins are generally absent from two‐dimensional gel proteome maps. In order to analyze the yeast plasma membrane proteome, a plasma membrane purification protocol was optimized in order to reduce contaminating membranes and cytosolic proteins. Specifically, the new fractionation scheme largely depleted the plasma membrane fraction of cytosolic proteins by deoxycholate stripping and ribosomal proteins by sucrose gradient flotation. The plasma membrane complement was resolved by two‐dimensional electrophoresis using the cationic detergent cetyl trimethyl ammonium bromide in the first, and sodium dodecyl sulfate in the second dimension, and fifty spots were identified by matrix‐assisted laser desorption/ionization‐time of flight mass spectometry. In spite of the presence of still contaminating ribosomal proteins, major proteins corresponded to known plasma membrane residents, the ABC transporters Pdr5p and Snq2p, the P‐type H+‐ATPase Pma1p, the glucose transporter Hxt7p, the seven transmembrane‐span Mrh1p, the low affinity Fe++ transporter Fet4p, the twelve‐span Ptr2p, and the plasma membrane anchored casein kinase Yck2p. The four transmembrane‐span proteins Sur7p and Nce102p were also present in the isolated plasma membranes, as well as the unknown protein Ygr266wp that probably contains a single transmembrane span. Thus, combining subcellular fractionation with adapted two‐dimensional electrophoresis resulted in the identification of intrinsic plasma membrane proteins.

Identification of peptidases in Nicotiana tabacum leaf intercellular fluid.

Peptidases in the extracellular space might affect the integrity of recombinant proteins expressed in, and secreted from, plant cells. To identify extracellular peptidases, we recovered the leaf intercellular fluid from Nicotiana tabacum plants by an infiltration–centrifugation method. The activity of various peptidases was detected by an in vitro assay in the presence of specific inhibitors, using BSA and human serum γ‐globulin as substrates. Peptidases were detected by 1‐ and 2‐D zymography in a polyacrylamide gel containing gelatin as substrate. Proteolytic activity was observed over a wide range of molecular masses equal to, or higher than, 45 kDa. To identify the peptidases, the extracellular proteins were digested with trypsin and analyzed by LC and MS. Seventeen peptides showing identity or similarity to predicted plant aspartic, cysteine, and serine peptidases have been identified. The extracellular localization of a cysteine peptidase aleurain homolog was also shown.

Different subcellular localization and glycosylation for a functional antibody expressed in Nicotiana tabacum plants and suspension cells.

Genes encoding the heavy and light chains of LO-BM2, a therapeutic IgG antibody, were assembled in the tandem or inverted convergent orientation and expressed in Nicotiana tabacum plants and BY-2 suspension cells. The tandem construct allowed higher expression in both expression systems. A similar degradation pattern was observed for the secreted antibody recovered from the leaf intercellular fluid and BY-2 culture medium. Degradation increased with leaf age or culture time. Antibodies purified from leaf tissues and BY-2 cells were both functional. However, MS analysis of the N-glycosylation showed complex plant-type glycans to be the major type in the antibody purified from plants, whereas, oligomannosidic was the major glycosylation type in that purified from BY-2 cells. LO-BM2 was observed mainly in the endoplasmic reticulum of BY-2 cells while, in leaf cells, it was localized mostly to vesicles resembling prevacuolar compartments. These results and those from endoglycosidase H studies suggest that LO-BM2 is secreted from BY-2 cells more readily than from leaf cells where it accumulates in a post-Golgi compartment.

Inactivation of the β(1,2)-xylosyltransferase and the α(1,3)-fucosyltransferase genes in Nicotiana tabacum BY-2 cells by a multiplex CRISPR/Cas9 strategy results in glycoproteins without plant-specific glycans

Plants or plant cells can be used to produce pharmacological glycoproteins such as antibodies or vaccines. However these proteins carry N-glycans with plant-typical residues [β(1,2)-xylose and core α(1,3)-fucose], which can greatly impact the immunogenicity, allergenicity, or activity of the protein. Two enzymes are responsible for the addition of plant-specific glycans: β(1,2)-xylosyltransferase (XylT) and α(1,3)-fucosyltransferase (FucT). Our aim consisted of knocking-out two XylT genes and four FucT genes (12 alleles altogether) in Nicotiana tabacum BY-2 suspension cells using CRISPR/Cas9. Three XylT and six FucT sgRNAs were designed to target conserved regions. After transformation of N. tabacum BY-2 cells with genes coding for sgRNAs, Cas9, and a selectable marker (bar), transgenic lines were obtained and their extracellular as well as intracellular protein complements were analyzed by Western blotting using antibodies recognizing β(1,2)-xylose and α(1,3)-fucose. Three lines showed a strong reduction of β(1,2)-xylose and α(1,3)-fucose, while two lines were completely devoid of them, indicating complete gene inactivation. The absence of these carbohydrates was confirmed by mass spectrometry analysis of the extracellular proteins. PCR amplification and sequencing of the targeted region indicated small INDEL and/or deletions between the target sites. The KO lines did not show any particular morphology and grew as the wild-type. One KO line was transformed with genes encoding a human IgG2 antibody. The IgG2 expression level was as high as in a control transformant which had not been glycoengineered. The IgG glycosylation profile determined by mass spectrometry confirmed that no β(1,2)-xylose or α(1,3)-fucose were present on the glycosylation moiety and that the dominant glycoform was the GnGn structure. These data represent an important step toward humanizing the glycosylation of pharmacological proteins expressed in N. tabacum BY-2 cells.

NtSCP1 from Tobacco Is an Extracellular Serine Carboxypeptidase III That Has an Impact on Cell Elongation

The leaf extracellular space contains several peptidases, most of which are of unknown function. We isolated cDNAs for two extracellular serine carboxypeptidase III genes from tobacco (Nicotiana tabacum), NtSCP1 and NtSCP2, belonging to a phylogenetic clade not yet functionally characterized in plants. NtSCP1 and NtSCP2 are orthologs derived from the two ancestors of tobacco. Reverse transcription-polymerase chain reaction analysis showed that NtSCP1 and NtSCP2 are expressed in root, stem, leaf, and flower tissues. Expression analysis of the β-glucuronidase reporter gene fused to the NtSCP1 transcription promoter region confirmed this expression profile. Western blotting of NtSCP1 and expression of an NtSCP1-green fluorescent protein fusion protein showed that the protein is located in the extracellular space of tobacco leaves and culture cells. Purified His-tagged NtSCP1 had carboxypeptidase activity in vitro. Transgenic tobacco plants overexpressing NtSCP1 showed a reduced flower length due to a decrease in cell size. Etiolated seedlings of these transgenic plants had shorter hypocotyls. These data provide support for a role of an extracellular type III carboxypeptidase in the control of cell elongation.

Expression and Secretion of Recombinant Outer-surface Protein A from the Lyme Disease Agent, Borrelia burgdorferi, in Nicotiana tabacum Suspension Cells

The ospA gene of Borrelia burgdorferi codes for an outer membrane lipoprotein, which is a major antigen of the Lyme disease agent. Recombinant OspA vaccines tested so far were expressed in Escherichia coli. In this study, we investigated the expression of a soluble OspA protein in Nicotiana tabacum suspension cells and evaluated the secretion of OspA driven by either its own bacterial signal peptide or a plant signal peptide fused to the amino-terminal cysteine of the mature form. In both cases, the signal peptide was cleaved off and OspA secreted. During secretion, OspA was N-glycosylated. Addition of a C-terminal KDEL sequence led to retention of OspA in the endoplasmic reticulum.