Recombinant human alpha 1-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form. This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature human AAT protein. Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide, and terminator from a rice alpha-amylase gene Amy3D. The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose. It was possible, therefore, to induce the expression of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino acids as those found in serum-derived (native) AAT from humans. This result indicates that the rice signal peptidase recognizes and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT. The highest molecular weight band seen on Western blots (AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity. Staining with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent than that observed with native AAT. The rAAT, purified by immunoaffinity chromatography, had the same association rate constant for porcine pancreatic elastase as the native AAT. Thermostability studies revealed that the rAAT and native AAT decayed at the same rate, suggesting that the rAAT is correctly folded. The productivity of rice suspension cells expressing rAAT was 4.6-5.7 mg/g dry cell. Taken together, these results support the use of rice cell culture as a promising new expression system for production of biologically active recombinant proteins.

The photosynthetic microalga Haematococcus pluvialis, a potential source of astaxanthin, was cultivated under illumination with LEDs emitting red (λmax= 625 nm), green (λmax= 525 nm), blue (λmax= 470 nm), blue-purple (λmax= 410 nm) and purple (λmax= 380 nm) light and a fluorescent lamp, and the effects of wavelength on cell growth and astaxanthin accumulation were studied. LEDs emitting light of short wavelengths (380 -470 nm) were found to induce astaxanthin accumulation of up to 5 -6% per dry-cell, although the induction caused the suppression of cell growth. From these results, we proposed a new strategy of cultivating H. pluvialis under illumination with red LEDs without inducing a high level of astaxanthin accumulation, and then switching to illumination with blue LEDs at a high light intensity to induce a high level of astaxanthin accumulation.

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