Production of a monoclonal antibody in plants with a humanized N-glycosylation pattern

In recent years, plants have become an attractive alternative for the production of recombinant proteins. However, their inability to perform authentic mammalian N -glycosylation may cause limitations for the production of therapeutics. A major concern is the presence of β1,2-xylose and core α1,3-fucose residues on complex N -linked glycans, as these N -glycan epitopes are immunogenic in mammals. In our attempts towards the humanization of plant N -glycans, we have generated an Arabidopsis thaliana knockout line that synthesizes complex N -glycans lacking immunogenic xylose and fucose epitopes. Here, we report the expression of a monoclonal antibody in these glycan-engineered plants that carry a homogeneous mammalian-like complex N -glycan pattern without β1,2-xylose and core α1,3-fucose. Plant and Chinese hamster ovary (CHO)-derived immunoglobulins (IgGs) exhibited no differences in electrophoretic mobility and enzyme-linked immunosorbent specificity assays. Our results demonstrate the feasibility of a knockout strategy for N -glycan engineering of plants towards mammalian-like structures, thus providing a significant improvement in the use of plants as an expression platform.     

Expression of the alpha3/beta1 isoform of human Na,K-ATPase in the methylotrophic yeast Pichia pastoris

Na,K-ATPase is a crucial enzyme for ion homeostasis in human tissues. Different isozymes are produced by assembly of four alpha- and three beta-subunits. The expression of the alpha3/beta1 isozyme is confined to brain and heart. Its heterologous production has so far never been attempted in a lower eukaryote. In this work we explored whether the methylotrophic yeast Pichia pastoris is capable of expressing the alpha3/beta1 isoform of human Na,K-ATPase. cDNAs encoding the alpha(3) and the beta(1)-subunits were cloned under the control of the inducible promoter of Pichia pastoris alcohol oxidase 1. Pichia pastoris could express the single alpha3- and beta1-subunits and even coexpress them after methanol induction. beta1-subunit was produced as a major 44-kDa glycosylated polypeptide and alpha3 as a 110-kDa unglycosylated polypeptide. Expression at the plasma membrane was limited in shaking flask cultures but by cultivating P. pastoris cells in a fermenter there was a 10-fold increase of the number of ouabain binding sites per cell. The exported enzyme was estimated to be about 0.230 mg L(-1) at the end of a bioreactor run. Na,K-ATPase proved active and the dissociation constant of the recombinant enzyme-ouabain interaction was determined.     

Maturation of rat brain is accompanied by differential expression of the long and short splice variants of G(s)alpha protein: identification of cytosolic forms of G(s)alpha

Distribution of the alpha subunit of the stimulatory G protein (G(s)alpha) was analyzed in membrane and cytosolic (supernatant 200 000 g) fractions from rat cortex, thalamus and hippocampus during the course of post-natal development. In parallel, changes in beta-adrenoceptor density and adenylyl cyclase activity were determined. Long (G(s)alphaL) and short (G(s)alphaS) variants of G(s)alpha were assessed by immunoblotting using specific polyclonal antisera reacting with both G(s)alpha isoforms. Post-natal development was associated with an increase in the total amount of brain G(s)alpha. G(s)alphaL was the dominant isoform of G(s)alpha in the membrane fractions of all studied brain regions and its amount increased markedly between post-natal day (PD) 1 and 90. The level of membrane-bound G(s)alphaS also elevated during post-natal development, but more pronounced changes were found in cytosolic G(s)alphaS. Although only a small amount of G(s)alphaS (much smaller than G(s)alphaL) was detected among soluble proteins shortly after birth, G(s)alphaS prevailed over G(s)alphaL at PD90. The G(s)alphaL/G(s)alphaS ratio decreased, respectively, from 3.2 to 1.2 and from 5.0 to 1.5 in the membrane fractions of cortex and hippocampus, but remained almost constant in thalamus between PD1 and 90. More dramatic changes were found in the cytosolic fractions of all studied brain regions: the G(s)alphaL/G(s)alphaS ratio decreased sharply in cortex (from 14.1 to 0.9), hippocampus (from 3.7 to 0.8), and also in thalamus (from 9.5 to 0.5). These results demonstrate that the membrane-cytosol balance of G(s)alpha proteins alters dramatically during the course of brain development. Both G(s)alphaL and G(s)alphaS were expressed in a region- and age-specific manner, which suggests different roles in the maturation of the brain tissue. A cyc(-) reconstitutive assay of cytosolic G(s)alpha indicated that only approximately 20% of this protein was functional, compared with membrane-bound G(s)alpha, and its ability to reconstitute adenylyl cyclase activity increased during the course of maturation. The number of beta-adrenoceptors increased sharply during early post-natal development but only slightly in adulthood, and both GTP- and isoproterenol-stimulated adenylate cyclase activity reached peak values around PD12.     

Expression of a novel member of the TGF-β superfamily, growth/differentiation factor-15/macrophage-inhibiting cytokine-1 (GDF-15/MIC-1) in adult rat tissues

We have cloned a novel member of the transforming growth factor-β (TGF-β) superfamily from a human placental cDNA library. The sequence is identical to five very recently published sequences, of which only one (macrophage inhibitory cytokine-1, MIC-1) has been characterized in terms of function. In light of the present data demonstrating the wide distribution of the mRNA and putative multifunctionality, we propose to name this molecule growth/differentiation factor-15/MIC-1 (GDF-15/MIC-1). The deduced amino acid sequence reveals typical features of a secreted molecule. The epithelium of the choroid plexus is the only site in the adult brain expressing detectable levels of GDF-15/MIC-1 mRNA. Many epithelia of non-neural tissues including those of the prostate and intestinal mucosa, bronchi and bronchioli, secretory tubuli of the submandibular gland, and lactating mammary gland are prominent sites of GDF-15/MIC-1 synthesis. GDF-15/MIC-1 is also strongly expressed by macrophages in the adrenal gland. Thus, GDF-15/MIC-1, like many other members of the TGF-β superfamily, is widely distributed in adult tissues, being most strongly expressed in epithelial cells and macrophages. Received: 25 January 1999 / Accepted: 17 March 1999