Expression of recombinant human acetylcholinesterase in transgenic tomato plants.

Enzyme therapy for the prevention and treatment of organophosphate poisoning depends on the availability of large amounts of cholinesterases. Transgenic plants are being evaluated for their efficiency and cost-effectiveness as a system for the bioproduction of therapeutically valuable proteins. Here we report production of a recombinant isoform of human acetylcholinesterase in transgenic tomato plants. Active and stable acetylcholinesterase, which retains the kinetic characteristics of the human enzyme, accumulated in tomato plants. High levels of specific activity were registered in leaves (up to 25 nmol min(-1) mg protein(-1)) and fruits (up to 250 nmol min(-1) mg protein(-1)).

Perspective: edible vaccines--a concept coming of age.

In addition to their traditional role as a source of natural medicines, it is now possible to genetically engineer plants to produce pharmaceuticals. Transgenic plants expressing antigens from pathogenic microorganisms offer many advantages as low-cost production systems and effective delivery systems for vaccines. This new technology might contribute to global vaccine programs and might have a dramatic impact on health care in developing countries.

An unusual organization of the genes encoding cytochrome b559 in Chlamydomonas reinhardtii: psbE and psbF genes are separately transcribed from different regions of the plastid chromosome.

The psbE and psbF genes encode the apoproteins of cytochrome b559, an essential component of the pigment protein complex photosystem II. Together with psbL and psbJ, these genes constitute a single operon in all photosynthetic organisms examined thus far. We have cloned and sequenced the psbE and psbF genes of the Chlamydomonas reinhardtii plastid genome. The predicted amino-terminal domains of both polypeptides are more basic than those of other organisms, and the sequence of the psbE gene product indicates a departure from the 'positive-inside' rule for the insertion of proteins in the thylakoid membrane. Northern blot analysis demonstrated that psbE is transcribed into a 0.3 kb mRNA, while transcription of psbF and psbL genes results in a 0.9 kb transcript. The splitting of the psbEFLJ operon into separate transcription units suggests a unique mechanism of regulation of expression of these genes in C. reinhardtii.

The ctpA gene encodes the C-terminal processing protease for the D1 protein of the photosystem II reaction center complex.

The D1 protein of the photosystem II (PSII) complex in the thylakoid membrane of oxygenic photosynthetic organisms is synthesized as a precursor polypeptide (pD1) with a C-terminal extension. Posttranslational processing of the pD1 protein is essential to establish water oxidation activity of the PSII complex. We have recently identified a gene, ctpA, a mutation in which resulted in a loss of PSII activity in the cyanobacterium Synechocystis sp. PCC 6803. To study the function of the CtpA protein, we inactivated the ctpA gene by inserting a kanamycin-resistance gene into its coding sequence. The resultant mutant strain, T564, had no PSII-mediated water oxidation activity, but it had normal cytochrome b6f and photosystem I activities. Measurements of thermoluminescence profiles and rates of reduction of 2,6-dichlorophenolindophenol indicated that PSII complexes in the mutant cells had functional reaction centers that were unable to accept electrons from water. Immunoblot analysis showed that D1, D2, CP47, CP43, and the alpha subunit of cytochrome b559, five integral membrane proteins of PSII, were present in T564 cells. Interestingly, the D1 protein in the mutant cells was 2 kDa larger than that in wild-type cells, due to the presence of a C-terminal extension. We conclude that the CtpA protein is a processing enzyme that cleaves off the C-terminal extension of the D1 protein. Interestingly, the CtpA protein shows significant sequence similarity to the interphotoreceptor retinoid-binding proteins in the bovine, human, and insect eye systems.