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.
Azzoni
Adriano R
AR
LEBp, Laboratório de Engenharia de Bioprocessos, Departamento de Processos Biotecnológicos, FEQ, UNICAMP, CP 6066, CEP 13083-970, Campinas, SP, Brazil.
Kusnadi
Ann R
AR
Miranda
Everson A
EA
Nikolov
Zivko L
ZL
D000327:Adsorption; D007611:Aprotinin / Q000235:genetics / Q000302:isolation & purification*; D002021:Buffers; D002846:Chromatography, Affinity / Q000379:methods; D003300:Copper / Q000737:chemistry; D018506:Gene Expression Regulation, Plant; D006863:Hydrogen-Ion Concentration; D010940:Plant Proteins / Q000302:isolation & purification; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*; D012639:Seeds / Q000737:chemistry* / Q000235:genetics; D012680:Sensitivity and Specificity; D012965:Sodium Chloride / Q000737:chemistry; D013696:Temperature; D003313:Zea mays / Q000737:chemistry / Q000235:genetics*
D002021:Buffers; D010940:Plant Proteins; D011994:Recombinant Proteins; C052160:trypsin inhibitor, Zea mays; D012965:Sodium Chloride; D003300:Copper; D007611:Aprotinin