Recombinant aprotinin produced in transgenic corn seed: extraction and purification studies.

Expression in transgenic plants is potentially one of the most economical systems for large-scale production of valuable peptide and protein products. However, the downstream processing of recombinant proteins produced in plants has not been extensively studied. In this work, we studied the extraction and purification of recombinant aprotinin, a protease inhibitor used as a therapeutic compound, produced in transgenic corn seed. Conditions for extraction from transgenic corn meal that maximize aprotinin concentration and its fraction of the total soluble protein in the extract were found: pH 3.0 and 200 mM NaCl. Aprotinin, together with a native corn trypsin inhibitor (CTI), was captured using a tryspin-agarose column. These two inhibitors were separated using an agarose-IDA-Cu2+ column that proved to efficiently absorb the CTI while the recombinant aprotinin was collected in the flowthrough with purity of at least 79%. The high purity of the recombinant aprotinin was verified by SDS-PAGE and N-terminal sequencing. The overall recombinant aprotinin recovery yield and purification factor were 49% and 280, respectively. Because CTI was also purified, the recovery and purification process studied has the advantage of possible CTI co-production. Finally, the work presented here introduces additional information on the recovery and purification of recombinant proteins produced in plants and corroborates with past research on the potential use of plants as biorreactors.

[1]  Rodrigo M. P. Siloto,et al.  Expression of correctly processed human growth hormone in seeds of transgenic tobacco plants , 2000, Molecular Breeding.

[2]  G. Zhong,et al.  Commercial production of aprotinin in transgenic maize seeds , 1999, Molecular Breeding.

[3]  V. Mackay,et al.  Nucleotide sequence of a cDNA clone that encodes the maize inhibitor of trypsin and activated Hageman factor , 1992, Plant Molecular Biology.

[4]  Gordon Allison,et al.  Transgenic plants as factories for biopharmaceuticals , 2000, Nature Biotechnology.

[5]  G. Nuttall,et al.  Plasma aprotinin concentrations during cardiac surgery: full- versus half-dose regimens. , 2000 .

[6]  E. A. Miranda,et al.  Recovery of aprotinin from insulin industrial process effluent by affinity adsorption , 1999 .

[7]  M. Vijayalakshmi,et al.  Aprotinin recovery: comparison between biospecific and pseudobiospecific affinity adsorptions , 1999 .

[8]  J. Boothe,et al.  Transgenic plants for therapeutic proteins: linking upstream and downstream strategies. , 1999, Current topics in microbiology and immunology.

[9]  C. Glatz,et al.  Process Engineering Strategy for Recombinant Protein Recovery from Canola by Cation Exchange Chromatography , 1999, Biotechnology progress.

[10]  E. Hood,et al.  Production and Purification of Two Recombinant Proteins from Transgenic Corn , 1998, Biotechnology progress.

[11]  J. Howard,et al.  Production of recombinant proteins in transgenic plants: Practical considerations , 1997, Biotechnology and bioengineering.

[12]  J. Suttnar,et al.  Separation used for purification of recombinant proteins. , 1997, Journal of chromatography. B, Biomedical sciences and applications.

[13]  C. Hollenberg,et al.  Production of two aprotinin variants in Hansenula polymorpha , 1996 .

[14]  C. Baufreton,et al.  Reduction of blood activation in patients receiving aprotinin during cardiopulmonary bypass for coronary artery surgery. , 1996, ASAIO journal.

[15]  R. J. Straub,et al.  An Overview of a Feasibility Study for the Production of Industrial Enzymes in Transgenic Alfalfaa , 1994, Annals of the New York Academy of Sciences.

[16]  M. Kula,et al.  Rapid purification of DesPro(2)‐Val15‐Leu17‐aprotinin from the culture broth of a recombinant Saccharomyces cerevisiae , 1993, Biotechnology and bioengineering.

[17]  G. Oshima.  Effects of different heparins on the enhancement of the thrombin-antithrombin reaction. , 1990, Biological chemistry Hoppe-Seyler.

[18]  E. Sulkowski.  The saga of IMAC and MIT. , 1989, BioEssays : news and reviews in molecular, cellular and developmental biology.

[19]  W. Schröder,et al.  Synthesis, cloning and expression of recombinant aprotinin. , 1987, Biological Chemistry Hoppe-Seyler.

[20]  G. Reeck,et al.  Combined use of trypsin-agarose affinity chromatography and reversed-phase high-performance liquid chromatography for the purification of single-chain protease inhibitor from corn seeds. , 1986, Journal of chromatography.

[21]  J J Pisano,et al.  Hageman factor fragment inhibitor in corn seeds: purification and characterization. , 1980, Thrombosis research.

[22]  G. Reeck,et al.  Isolation and characterization of trypsin inhibitor from opaque-2 corn seeds. , 1977, The Journal of biological chemistry.

[23]  B. Kassell.  [66b] Bovine trypsin-kallikrein inhibitor (kunits inhibitor, basic pancreatic trypsin inhibitor, polyvalent inhibitor from bovine organs) , 1970 .

[24]  J. Northrop,et al.  ISOLATION FROM BEEF PANCREAS OF CRYSTALLINE TRYPSINOGEN, TRYPSIN, A TRYPSIN INHIBITOR, AND AN INHIBITOR-TRYPSIN COMPOUND , 1936, The Journal of general physiology.