Production of functional human alpha(1)-antitrypsin by plant cell culture

Recombinant human al-antitrypsin (rAAT) was expressed and secreted from tra nsgenic rice cell suspension cultures in its biologically active form. This was accomplished by transforming: rice callus tissues with an expression v ector, p3D-AAT, containing the cDNA for mature human AAT protein. Regulated expression and secretion of rAAT from this vector was achieved using the p romoter, signal peptide, and terminator from a rice alpha-amylase gene Amy3 D. The Amy3D gene of rice is tightly controlled by simple sugars such as su crose. 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 prod uced a heterogeneous population of the rAAT molecules, they had the same N- terminal amino acids as those found in serum-derived (native) AAT from huma ns. This result indicates that the rice signal peptidase recognizes and cle aves the novel sequence between the Amy3D signal peptide and the first amin o acid of the mature human AAT. The highest molecular weight band seen on W estern blots (AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity. Staining with biotin-co ncanavalin 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 associati on rate constant for porcine pancreatic elastase as the native AAT. Thermos tability studies revealed that the rAAT and native AAT decayed at the same rate, suggesting that the rAAT is correctly folded. The productivity of ric e suspension cells expressing rAAT was 4.6-5.7 mg/g dry cell. Taken togethe r, these results support the use of rice cell culture as a promising new ex pression system for production of biologically active recombinant proteins.