High Recovery Refolding of rhG-CSF from Escherichia coli, Using Urea Gradient Size Exclusion Chromatography

Abstract

Protein folding liquid chromatography (PFLC) is a powerful tool for simultaneous refolding and purification of recombinant proteins in inclusion bodies. Urea gradient size exclusion chromatography (SEC) is a recently developed protein refolding method based on the SEC refolding principle. In the presented work, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escheriachia coli (E. coli) in the form of inclusion bodies was refolded with high yields by this method. Denatured/reduced rhG-CSF in 8.0 mol‚L -1 urea was directly injected into a Superdex 75 column, and with the running of the linear urea concentration program, urea concentration in the mobile phase and around the denatured rhG-CSF molecules was decreased linearly, and the denatured rhG-CSF was gradually refolded into its native state. Aggregates were greatly suppressed and rhG-CSF was also partially purified during the refolding process. Effects of the length and the final urea concentration of the urea gradient on the refolding yield of rhG-CSF by using urea gradient SEC were investigated respectively. Compared with dilution refolding and normal SEC with a fixed urea concentration in the mobile phase, urea gradient SEC was more efficient for rhG-CSF refoldingsin terms of specific bioactivity and mass recovery, the denatured rhG-CSF could be refolded at a larger loading volume, and the aggregates could be suppressed more efficiently. When 500 µL of solubilized and denatured rhG-CSF in 8.0 mol‚L -1 urea solution with a total protein concentration of 2.3 mg‚mL -1 was loaded onto the SEC column, rhG-CSF with a specific bioactivity of 1.0 × 10 8 IU‚mg -1 was obtained, and the mass recovery was 46.1%.

High pH Solubilization and Chromatography-Based Renaturation and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor from Inclusion Bodies

“…Previously we refolded rhG-CSF, which was solubilized by 8 M urea, by using various liquid chromatographic methods [13,[23][24][25][26]. Though higher refolding yields were approached when compared to dilution refolding method, the highest mass recovery in these work is only 49% [24].…”

“…As one of matrix-assisted refolding methods, protein refolding by liquid chromatography can accomplish refolding and purification simultaneously and has been applied on many proteins [10,11]. There are mainly four kinds of liquid chromatography which have been used to protein refolding: size exclusion chromatography [12,13], affinity chromatography [14][15][16], ion exchange chromatography [17][18][19], and hydrophobic interaction chromatography [20][21][22].…”

“…When rhG-CSF is produced in E. coli, it often forms inclusion bodies, thus solubilization and subsequent refolding are necessary to recover the useful and bioactive protein. We have extensively investigated the refolding and purification of urea-solubilized rhG-CSF from inclusion bodies by using various chromatography-based methods [13,[23][24][25][26]. However, the protein yield is still anticipated to be enhanced.…”

Urea-Gradient Protein Refolding in Size Exclusion Chromatography

“…While a suitable urea concentration used as a column elution buffer is important for normal SEC refolding [7,9], the final urea concentration is critical for UGSEC. The final urea concentration for UGSEC is the optimal urea concentration for normal SEC refolding, as has been the case for lysozyme [7] and rhG-CSF [9].…”

Refolding of scFv mini-antibodies using size-exclusion chromatography via arginine solution layer

“…However, even under these conditions correct complete protein folding does not take place always, and sometimes an aggregation is observed. To avoid an aggregation, low concentrations of denaturants such as 0.6 M guanidine hydrochloride [11,12] or 1-2.5 M urea [13,14] are maintained in renaturation medium. At the same time it is widely known that arginine, when added at intermediate and/or final steps of protein renaturation, facilitates its folding and stabilization of its native conformations.…”