Production of functional human α1-antitrypsin by plant cell culture

M. Terashima, Y. Murai, M. Kawamura, S. Nakanishi, T. Stoltz, L. Chen, W. Drohan, R. L. Rodriguez, S. Katoh

Research output: Contribution to journalArticle

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Abstract

Recombinant human α1-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 α-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.

Original languageEnglish (US)
Pages (from-to)516-523
Number of pages8
JournalApplied Microbiology and Biotechnology
Volume52
Issue number4
DOIs
StatePublished - 1999

Fingerprint

Plant cell culture
Plant Cells
Sugar (sucrose)
Sucrose
Suspensions
Cell Culture Techniques
Pancreatic Elastase
Protein Sorting Signals
Amino acids
Amino Acids
Genes
Glycosylation
Avidin
Horseradish Peroxidase
Amylases
Biotin
Concanavalin A
Chromatography
Cell culture
Recombinant Proteins

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology
  • Bioengineering

Cite this

Terashima, M., Murai, Y., Kawamura, M., Nakanishi, S., Stoltz, T., Chen, L., ... Katoh, S. (1999). Production of functional human α1-antitrypsin by plant cell culture. Applied Microbiology and Biotechnology, 52(4), 516-523. https://doi.org/10.1007/s002530051554

Production of functional human α1-antitrypsin by plant cell culture. / Terashima, M.; Murai, Y.; Kawamura, M.; Nakanishi, S.; Stoltz, T.; Chen, L.; Drohan, W.; Rodriguez, R. L.; Katoh, S.

In: Applied Microbiology and Biotechnology, Vol. 52, No. 4, 1999, p. 516-523.

Research output: Contribution to journalArticle

Terashima, M, Murai, Y, Kawamura, M, Nakanishi, S, Stoltz, T, Chen, L, Drohan, W, Rodriguez, RL & Katoh, S 1999, 'Production of functional human α1-antitrypsin by plant cell culture', Applied Microbiology and Biotechnology, vol. 52, no. 4, pp. 516-523. https://doi.org/10.1007/s002530051554
Terashima, M. ; Murai, Y. ; Kawamura, M. ; Nakanishi, S. ; Stoltz, T. ; Chen, L. ; Drohan, W. ; Rodriguez, R. L. ; Katoh, S. / Production of functional human α1-antitrypsin by plant cell culture. In: Applied Microbiology and Biotechnology. 1999 ; Vol. 52, No. 4. pp. 516-523.
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