A simplified method for the efficient refolding and purification of recombinant human GM-CSF

Abstract

Human granulocyte macrophage colony-stimulating factor (hGM-CSF) is a haematopoietic growth factor and proinflammatory cytokine. Recombinant hGM-CSF is important not only as a research tool but also as a biotherapeutic. However, rhGM-CSF expressed in E. coli is known to form inclusion bodies of misfolded, aggregated protein. Refolding and subsequent purification of rhGM-CSF from inclusion bodies is difficult with low yields of bioactive protein being produced. Here we describe a method for the isolation, refolding and purification of bioactive rhGM-CSF from inclusion bodies. The method is straightforward, not requiring extensive experience in protein refolding nor purification and using standard laboratory equipment.

Figure 1. rhGM-CSF forms inclusion bodies when expressed in E. coli.

(A) Shown is the 15% reducing SDS PAGE analysis of the expression of rhGM-CSF transformed into the BL21(DE3) E. coli strain. Upon addition of IPTG (+) the cells efficiently express the rhGM-CSF protein. (B) Following induction with IPTG, the bacteria were lysed by mechanical disruption and the cell supernatant separated from the pellet by centrifugation. Shown is the 15% reducing SDS PAGE analysis of the cell lysate prior to centrifugation, and the subsequent centrifugal supernatant and pellet fractions. The molecular weight marker is PageRuler Prestained Protein Ladder from Fermentas Life Sciences and the gels are stained with Coomassie Brilliant Blue.

Figure 2. rhGM-CSF can be purified following refolding from inclusion bodies and His-tag affinity chromatography.

Shown is the 15% reducing SDS PAGE analysis of purified rhGM-CSF following His-tag affinity chromatography and low salt dialysis. The molecular weight marker is PageRuler Prestained Protein Ladder from Fermentas Life Sciences and the gel is stained with Coomassie Brilliant Blue.

Figure 3. Mass spectral analysis of purified rhGM-CSF.

(A) Shown is the sequence of the expressed rhGM-CSF. In bold and underlined is rhGM-CSF sequence coverage obtained by the mass spectral analysis. 95% sequence coverage was obtained, not including the 8×His tag. The rhGM-CSF was cleaved separately by trypsin and Protease V8 and the generated peptides likewise analyzed separately by LC-MS/MS (FT-ICR). (B) The MS/MS spectrum of the doubly charged peptide MFDLQEPTCLQTRLE (m/z 948.95), which is the fragment of protein granulocyte-macrophage colony-stimulating factor (amino acid residues 63–77) digested by protease V8, is shown as a representative spectra. M: Methionine oxidation; C: Cystine carbamidomethyl modification.

Figure 4. Purified rhGM-CSF demonstrates bioactivity.

Shown is the proliferation of TF-1 cells after incubation for 4 days in the presence of purified rhGM-CSF. For comparison, a commercial source of rhGM-CSF was also analyzed. TF-1 cells require the presence of GM-CSF for survival and therefore their proliferation with the addition of GM-CSF, as measured using the WST-1 reagent, is a measure of GM-CSF bioactivity. Absorbance values are proportional to the number of viable cells. Data are the mean ±SD of triplicate measurements.