Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine.
Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants.
Platis
Dimitris
D
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Athens, Greece.
Labrou
Nikolaos E
NE
D000327:Adsorption; D000818:Animals; D016466:CHO Cells; D002417:Cattle; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000737:chemistry / Q000302:isolation & purification; D006224:Cricetinae; D003412:Cricetulus; D006632:Histamine / Q000378:metabolism; D006801:Humans; D008024:Ligands; D018515:Plant Leaves / Q000737:chemistry / Q000235:genetics; D030821:Plants, Genetically Modified / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000627:therapeutic use; D014026:Nicotiana; D003313:Zea mays / Q000737:chemistry / Q000235:genetics*
D008024:Ligands; D011994:Recombinant Proteins; D006632:Histamine; D002918:Chymotrypsin; C117608:alpha-chymotrypsin