Negative chromatography on agarose-TREN as a technique for purification of protein spiked in soybean seeds extract

• Published in: Process biochemistry (1991) 2012-12, Vol.47 (12), p.2255-2261
• Main Authors: Bresolin, Iara Rocha Antunes Pereira, Bresolin, Igor Tadeu Lazzarotto, Miranda, Everson Alves, Bueno, Sonia Maria Alves
• Format: Article
• Language: English
• Subjects: adsorbents; adsorption; chromatography; Downstream processing; electrostatic interactions; human serum albumin; humans; hydrophobicity; immunoglobulin G; ligands; Mixed-mode chromatography; Negative chromatography; polyamines; purification methods; recombinant proteins; seeds; soy protein; Soybean extract; soybeans; transgenic plants; Tris(2-aminoethyl)amine (TREN)
• ISSN: 1359-5113; 1873-3298
• DOI: 10.1016/j.procbio.2012.08.023

► Proteins extracted from soybean were extensively adsorbed onto agarose-TREN. ► Human IgG was obtained in nonretained chromatographic fractions (negative chromatography). ► The predominance of electrostatic interactions between immobilized TREN and proteins was verified. ► Dilution of the soybean extract is necessary to avoid precipitation of proteins in the column. Alkyl amines and polyamines have been used as ligands for protein purification by mixed-mode chromatography. The adsorption of proteins onto these ligands seems to be governed by multiple effects such as electrostatic, hydrophobic, and affinity interactions. In this work we investigated the adsorption of proteins extracted from soybean onto the adsorbent agarose-Tris(2-aminoethyl)amine (TREN). The effects of flow rate, buffer system, and extract concentration on the capture of proteins extracted from soybean were evaluated. Experiments using Mes at pH 6.5 as adsorption buffer allowed the adsorption of almost the totality of native soybean protein with a dynamic adsorption capacity of 13.50mgmL−1 adsorbent. Experiments with human IgG (pI in the range of 5.8–9.0) and human serum albumin (HSA, pI of 4.9) spiked into these extracts lead to the conclusion that electrostatic forces play a major role in the interaction between protein and agarose-TREN. Based on this work, negative chromatography with agarose-TREN should be considered as a method for purification of basic recombinant protein produced in transgenic soybean seeds.