Transient expression in tobacco leaves of an aglycosylated recombinant antibody against the epidermal growth factor receptor.

When generating stably transformed transgenic plants, transient gene expression experiments are especially useful to rapidly confirm that the foreign molecule of interest is correctly assembled and retains its biological activity. TheraCIM(R) (CIMAB S.A., Havana) is a recombinant humanized antibody against the Epidermal Growth Factor receptor (EGF-R), now in clinical trials for cancer therapy in Cuba and other countries. An aglycosylated version (Asn 297 was mutated for Gln 297) of this antibody was transiently expressed in tobacco leaves after vacuum-mediated infiltration of recombinant Agrobacterium tumefaciens that carried a binary plasmid bearing the antibody heavy and light chain genes and plant regulation signals. Protein extracts from "agroinfiltrated" leaves were tested by ELISA and Western blot, showing that the fully assembled antibody was accumulated in plant tissues. The absence of plant specific glycans did not interfere in the assembling or in the activity of the plantibody, as demonstrated in this work. Indirect immunofluorescence demonstrated that the aglycosylated antibody expressed in plants recognizes the EGF-R expressed on the surface of A431 human tumor culture cells.

Expression and characterization of an anti-(hepatitis B surface antigen) glycosylated mouse antibody in transgenic tobacco (Nicotiana tabacum) plants and its use in the immunopurification of its target antigen.

Transgenic plants expressing recombinant immunoglobulins have arisen as an alternative technology for the large-scale production of antibodies useful in therapeutics and in industrial processes. In the present paper we report the expression in transgenic tobacco ( Nicotiana tabacum ) of an anti-HBsAg [anti-(hepatitis B virus surface antigen)] mouse IgG1 mAb (monoclonal antibody), currently used for the industrial purification of the recombinant vaccine antigen. Using the sweet potato sporamin signal peptide, a KDEL (Lys-Asp-Glu-Leu) ER (endoplasmic reticulum) anchorage domain, and a heavy- and light-chain gene tandem construction, we generated F1 plants in which the expression of the antibody accounted for 0.5% of the total soluble proteins. The 'plantibody' (functional IgG antibody produced in plants) was easily purified by Protein A-Sepharose chromatography with a yield of approximately 35 microg/g of fresh leaf material, and its glycosylation indicated that, irrespective of the KDEL signal, the molecule is modified in both the ER and Golgi. Finally, a successful comparison of the plantibody with the ascites-derived mAb in the immunoaffinity purification of the vaccine recombinant HBsAg was performed. Taken as a whole, our results show that the large-scale production of this antibody of industrial relevance in transgenic tobacco is feasible.

Efecto de diferentes métodos de cultivo sobre la regeneración in vitro de papa (Solanum tuberosum L.) de la vaeriedad comerciall Chieftain

Se evaluó la capacidad de regeneración de explantes de hoja, tubérculos y segmentos de tallo de la variedad comercial Chieftan, utilizando dos métodos: A, el mismo medio de cultivo desde la iniciación de los callos hasta la formación de los brotes y B, transferencia de los callos a un medio de cultivo libre de auxinas antes de la formación de los brotes. La mayor frecuencia de regeneración se obtuvo de discos de tubérculo en el medio (MRT-6) (método A) con 3,28 brotes por explante, seguido por 2,5 brotes en segmentos de tallo (medios MRE-4 y MRE-6, método B) y 0,8 brotes a partir de discos de hoja (medio MRH-3, método A). La combinación de 1 mg/L de nitrato de plata con 5 g/L de carbón activado, durante la propagación in vitro, mostró diferencias significativas tanto en el número de hojas por planta como en su calidad (AU)

Genetic transformation of sugarcane by agrobacterium tumefaciens using antioxidant compounds

Se evaluaron los efectos de tres compuestos antioxidantes - ácido ascórbico, cisteína y nitrato de plata - sobre el crecimiento de Agrobacterium cepa At 2260 y la interacción de éste con los explantes vegetales, así como sobre la viabilidad y la formación de callos de tejidos meristemáticos de caña de azúcar variedad comercial Ja 60-5, provenientes de plantas in vitro y de campo. Se logró la transferencia de un vector binario con los genes uid1 y bar a explanter meristemáticos de caña de azúcar y se obtuvieron callos resistentes a BASTA y GUS-positivos. El empleo de la mezcla de antioxidantes redujo en un 80 porciento la muerte celular con relación al control, y no alteró la calidad del callo en ninguna de las fases de cultivo (AU)

Molecular characterization of the levansucrase gene from the endophytic sugarcane bacterium Acetobacter diazotrophicus SRT4.

The Acetobacter diazotrophicus SRT4 gene encoding levansucrase (EC 2.4.1.10) (IsdA) was isolated from a genomic library. The nucleotide sequence of a 2.3 kb DNA fragment sufficient for complementation of a levansucrase-deficient mutant (obtained by EMS treatment) was determined. The IsdA gene (1751 bp) coded for a polypeptide of molecular mass 64.9 kDa with an isoelectric point of 5.2. The N-terminal amino acid sequence of the extracellular levansucrase indicated the presence of a precursor protein with a putative signal sequence of 51 residues which is possibly cleaved in two successive steps. Expression of the IsdA gene from the lac promoter in Escherichia coli resulted in the production of a protein with levansucrase activity. The deduced amino acid sequence of the IsdA gene was 48% and 46% identical with the levansucrases from the Gram-negative bacteria Zymomonas mobilis and Erwinia amylovora, respectively, but only 28-31% identical with levansucrases from Gram-positive bacteria. Multiple alignments of published levansucrase sequences from Gram-negative and Gram-positive bacteria revealed eight conserved motifs. A comparison of the catalytic properties and the sequence of the A. diazotrophicus levansucrase with those of the Bacillus subtilis levansucrase suggested that one of these motifs may be involved in the specificity of the synthetized product. Disruption of the IsdA gene in the genome of A. diazotrophicus resulted in a mutant lacking both levansucrase activity and the ability to utilize sucrose as a carbon source, suggesting that levansucrase is the key enzyme in sucrose metabolism of A. diazotrophicus.