Separation of human Fab fragments on negative mode Ni(II)-TREN-agarose chromatography

•Human Fab fragments were obtained in nonretained chromatographic fractions (negative chromatography).•Proteins extracted from soybean seeds were adsorbed on Ni(II)-TREN-agarose.•The selectivity of the Ni(II)-TREN-agarose was influenced by the nature of the buffer system and NaCl. We evaluated the f...

Full description

Saved in:
Bibliographic Details
Published in:Process biochemistry (1991) 2014-04, Vol.49 (4), p.715-723
Main Authors: da Silva, Luana Cristina Andrade, Serracchiani, Marcel Mafei, Miranda, Everson Alves, Bueno, Sonia Maria Alves
Format: Article
Language:English
Subjects:
Chromatography
Fragments
Human
Human Fab fragments
IMAC
Ni(II)-TREN
Proteins
Purification
Purity
Soybean proteins
Soybeans
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:•Human Fab fragments were obtained in nonretained chromatographic fractions (negative chromatography).•Proteins extracted from soybean seeds were adsorbed on Ni(II)-TREN-agarose.•The selectivity of the Ni(II)-TREN-agarose was influenced by the nature of the buffer system and NaCl. We evaluated the feasibility of using immobilized metal-ion affinity chromatography (IMAC) with nickel ion complexed with Tris(2-aminoethyl)amine (TREN) immobilized on agarose gel for purification of human Fab fragments by negative chromatography. Efficient purification of Fab fragments from digested human IgG (immunoglobulin G) (106.4% purity) was accomplished in Tris-HCl buffer at pH 7.5 without NaCl (based on total protein concentration and radial immunodiffusion of human Fab). A technological application of Ni(II)-TREN-agarose using non transgenic soybean protein extract spiked with human Fab fragments as feedstream was also studied. Experiments using Tris-HCl at pH 7.0 as loading buffer allowed the adsorption of almost all of the soybean proteins. Sixty-six percent of the loaded human Fab fragments were recovered in the flowthrough and washing fractions with about 90% purity. These results demonstrate that Ni(II)-TREN-agarose is a potential adsorbent for recombinant Fab fragment purification.
ISSN:1359-5113
1873-3298
DOI:10.1016/j.procbio.2013.12.006