Biomedical subjects

J W Molthoff

Publications and source records attributed to J W Molthoff.

6 recordsLinked to original sources

PubMed2001

Oral immunisation of naive and primed animals with transgenic potato tubers expressing LT-B.

The efficacy of edible vaccines produced in potato tubers was examined in mice. Transgenic plants were developed by Agrobacterium tumefaciens-mediated transformation. The antigen selected was the non-toxic B subunit of the Escherichia coli enterotoxin (recLT-B). A synthetic gene coding for recLT-B was made and optimised for expression in potato tubers and accumulation in the endoplasmic reticulum. Introduction of this gene under control of the tuber-specific patatin promoter in potato plants resulted in the production of functional, i.e. Gm1-binding, recLT-B pentamers in tubers. Selected tubers containing about 13 microg of recLT-B per gram fresh weight were used for immunisation. Subcutaneous immunisation with an extract of recLT-B tubers yielded high antibody titres in serum that were similar to those obtained with bacterial recLT-B. The efficacy of oral administration of recLT-B tubers was determined by measuring mucosal and systemic immune responses in naive and primed mice. Animals were primed by subcutaneous injection of an extract of recLT-B tuber plus adjuvant. Naive and primed mice were fed 5 g of tubers ( approximately 65 microg of recLT-B) or were intubated intragastrically with 0.4 ml of tuber extract ( approximately 2 microg of recLT-B). In naive mice, feeding recLT-B tubers or intubation of tuber extract did not induce detectable anti-LT antibody titres. In primed animals, however, oral immunisation resulted in significant anti-LT IgA antibody responses in serum and faeces. Intragastric intubation of tuber extract revealed higher responses than feeding of tubers. These results indicate clearly that functional recLT-B can be produced in potato tubers, that this recombinant protein is immunogenic and that oral administration thereof elicits both systemic and local IgA responses in parentally primed, but not naive, animals.

Administration, Oral↗

PubMed2001

Galactose-extended glycans of antibodies produced by transgenic plants.

Plant-specific N-glycosylation can represent an important limitation for the use of recombinant glycoproteins of mammalian origin produced by transgenic plants. Comparison of plant and mammalian N-glycan biosynthesis indicates that beta1,4-galactosyltransferase is the most important enzyme that is missing for conversion of typical plant N-glycans into mammalian-like N-glycans. Here, the stable expression of human beta1,4-galactosyltransferase in tobacco plants is described. Proteins isolated from transgenic tobacco plants expressing the mammalian enzyme bear N-glycans, of which about 15% exhibit terminal beta1,4-galactose residues in addition to the specific plant N-glycan epitopes. The results indicate that the human enzyme is fully functional and localizes correctly in the Golgi apparatus. Despite the fact that through the modified glycosylation machinery numerous proteins have acquired unusual N-glycans with terminal beta1,4-galactose residues, no obvious changes in the physiology of the transgenic plants are observed, and the feature is inheritable. The crossing of a tobacco plant expressing human beta1,4-galactosyltransferase with a plant expressing the heavy and light chains of a mouse antibody results in the expression of a plantibody that exhibits partially galactosylated N-glycans (30%), which is approximately as abundant as when the same antibody is produced by hybridoma cells. These results are a major step in the in planta engineering of the N-glycosylation of recombinant antibodies.

Antibodies↗

PubMed2001

Influence of growth conditions and developmental stage on N-glycan heterogeneity of transgenic immunoglobulin G and endogenous proteins in tobacco leaves.

Plants are regarded as a promising system for the production of heterologous proteins. However, little is known about the influence of plant development and growth conditions on N-linked glycosylation. To investigate this, transgenic tobacco (Nicotiana tabacum cv Samsun NN) plants expressing a mouse immunoglobulin G antibody (MGR48) were grown in climate rooms under four different climate conditions, i.e. at 15 degrees C and 25 degrees C and at either low or high light conditions. N-glycans on plantibodies and soluble endogenous proteins were analyzed with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). Antibodies isolated from young leaves have a relatively high amount of high- mannose glycans compared with antibodies from older leaves, which contain more terminal N-acetylglucosamine. Senescence was shown to affect the glycosylation profile of endogenous proteins. The relative amount of N-glycans without terminal N-acetylglucosamine increased with leaf age. Major differences were observed between glycan structures on endogenous proteins versus those on antibodies, probably to be attributed to their subcellular localization. The relatively high percentage of antibody N-glycan lacking both xylose and fucose is interesting.

Animals↗

PubMed2000

Effect of climate conditions and plant developmental stage on the stability of antibodies expressed in transgenic tobacco.

Plants are regarded as a promising system for the production of heterologous proteins. However, little is known about the influence of plant physiology and plant development on the yield and quality of the heterologous proteins produced in plants. To investigate this, tobacco (Nicotiana tabacum cv Samsun NN) was transformed with a single construct that contained behind constitutive promotors the light- and heavy-chain genes of a mouse antibody. The in planta stability of the antibody was analyzed in transgenic plants that were grown under high and low irradiation at 15 degrees C and 25 degrees C. High-light conditions favored the production of biomass, of total soluble protein, and of antibody. The plants grown at 25 degrees C developed faster and contained less antibody per amount of leaf tissue than the plants grown at 15 degrees C. Both endogenous protein and antibody content showed a strong decline during leaf development. The heavy chains of the antibody underwent in planta degradation via relatively stable fragments. In vitro incubations of purified plantibody with leaf extracts of wild-type tobacco indicated the involvement of acidic proteases. It is interesting that the same antibody produced by mouse hybridoma cells exhibited higher stability in this in vitro assay. This may be explained by the assumption that the plant type of N-glycosylation contributes less to the stability of the antibody than the mouse-type of N-glycosylation. The results of this study indicate that proteolytic degradation during plant development can be an important factor affecting yield and homogeneity of heterologous protein produced by transgenic plants.

Animals↗

PubMed1995

pBINPLUS: an improved plant transformation vector based on pBIN19.

We describe the construction of a new plant transformation vector, pBINPLUS, based on the popular pBIN19 vector. Improvements over pBIN19 include location of the selectable marker gene at the left T-DNA border, a higher copy number in E. coli, and two rare restriction sites around the multiple cloning site for easier cloning and analysis of T-DNA insertions in plant genomes.

Agrobacterium tumefaciens↗

PubMed1994

Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roots of transgenic tobacco.

To explore the feasibility of employing antibodies to obtain disease resistance against plant root pathogens, we have studied the expression of genes encoding antibodies in roots of transgenic plants. A model monoclonal antibody was used that binds to a fungal cutinase. Heavy and light chain cDNAs were amplified by PCR, fused to a signal sequence for secretion and cloned behind CaMV 35S and TR2' promoters in a single T-DNA. The chimeric genes were cloned both in tandem and in a divergent orientation. The roots of tobacco plants transformed with these constructs produced antibodies that were able to bind antigen in an ELISA. Immunoblotting showed assembly to a full-size antibody. In addition, a F(ab')2-like fragment was observed, which is probably formed by proteolytic processing. Both antibody species were properly targeted to the apoplast, but the full-size antibody was partially retained by the wall of suspension cells. The construct with divergent promoters showed a better performance than the construct with promoters in tandem. It directed the accumulation of functional antibodies to a maximum of 1.1% of total soluble protein, with half of the plants having levels higher than 0.35%. The high efficiency of this construct probably results from coordinated and balanced expression of light and heavy chain genes, as evidenced by RNA blot hybridization.

Amino Acid Sequence↗