Single-step purification of recombinant green fluorescent protein on expanded beds of immobilized metal affinity chromatography media

• Published in: Biochemical engineering journal Vol. 42; no. 3; pp. 301 - 307
• Main Authors: Dalal, Sohel, Raghava, Smita, Gupta, M.N.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01-12-2008
• Subjects: Adsorption; Bioseparation; Fluidization; Green fluorescent protein; Immobilized metal affinity chromatography; Purification; Immobilized metal affinity chromatography; Purification; Fluidization; Adsorption; Green fluorescent protein; Bioseparation
• ISSN: 1369-703X; 1873-295X
• DOI: 10.1016/j.bej.2008.07.010

Immobilized metal ion affinity chromatography (IMAC) in expanded bed mode is used for purifying recombinant green fluorescent protein (GFP) overexpressed in
Escherichia coli. The purification is carried out on two different matrices, i.e. Ni
2+ Streamline™ and Ni
2+ cross-linked alginate beads. The binding isotherms to both IMAC media followed the Langmuir model. The maximum binding capacity (
q
max) of Ni
2+ Streamline™ and Ni
2+ cross-linked alginate for the GFP was 1,42,860
FU
ml
−1 and 18,000
FU
ml
−1, respectively. The expanded bed column chromatography using Ni
2+ Streamline™ gave 2.7-fold purification with 89% of GFP recovery, while Ni
2+ alginate gave 3.1-fold purification with 91% of GFP recovery. SDS-PAGE of purified GFP in both cases showed single band. The results obtained in the expanded bed chromatography are compared with those obtained in packed bed chromatography.