Purification and Refolding of Overexpressed Human Basic Fibroblast Growth Factor inEscherichia coli

Hasan Mirzahoseini,Mona Alibolandi

doi:10.4061/2011/973741

Hasan Mirzahoseini, Mona Alibolandi

Open Access

https://doi.org/10.4061/2011/973741

Copy DOI

Abstract

This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubilization in 8 M urea. The adsorption was made in expanded bed without clarification steps such as centrifugation. Column refolding was done by elimination of urea and elution with NaCl. The human basic fibroblast growth factor was obtained as a highly purified soluble monomer form with similar behavior in circular dichroism and fluorescence spectroscopy as native protein. A total of 92.52% of the available human basic fibroblast growth factor was recovered as biologically active and purified protein using the mentioned purification and refolding process. This resulted in the first procedure describing high-throughput purification and refolding of human basic fibroblast growth factor in one step and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution.

Highlights

Production of therapeutic proteins in inclusion bodies is useful due to the efficacy of insoluble expression such as high product yield and protection against degradation by proteases [1]
The methods used for inclusion body solubilization result in a soluble protein that is biologically inactive
Heparin affinity chromatography is commonly combined with a cation-exchange step, maybe followed from subsequent purification steps, such as copper chelate or hydrophobic interaction or gel permeation chromatography [9, 10]

Summary

Introduction

Production of therapeutic proteins in inclusion bodies is useful due to the efficacy of insoluble expression such as high product yield and protection against degradation by proteases [1]. Heparin affinity chromatography is commonly combined with a cation-exchange step, maybe followed from subsequent purification steps, such as copper chelate or hydrophobic interaction or gel permeation chromatography [9, 10]. These traditional techniques are limited with recombinant proteins by intrinsic problem of aggregation [11]. The challenge is to convert the inactive and insoluble inclusion bodies and small protein aggregates into soluble, correctly folded biologically active products [12]. The cell homogenate including inclusion bodies and small soluble aggregates was dissolved by urea and the unfolded recombinant hbFGF was allowed to adsorb on the chromatographic support. The incentive of this approach was to obtain a one step purification of high quantity of hbFGF suitable for automation

Materials and Methods

Chromatographic Procedure

Analytical Procedures

Results and Discussion