Ly express the rhGM-CSF protein. (B) Following induction with IPTG, the

Ly 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. doi:10.1371/journal.pone.0049891.gPurification of Recombinant Human GM-CSFFigure 3. Mass spectral analysis of MedChemExpress Madrasin Purified rhGM-CSF. (A) Shown is the sequence of the expressed rhGM-CSF. In bold and underlined is rhGMCSF sequence coverage obtained by the 18334597 mass spectral analysis. 95 sequence coverage was obtained, not including the 86His 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 colonystimulating factor (amino acid residues 63?7) digested by protease V8, is shown as a representative spectra. M: Methionine oxidation; C: Cystine carbamidomethyl modification. doi:10.1371/journal.pone.0049891.gTools was performed. The titrations of purified rhGM-CSF or the commercial MedChemExpress Docosahexaenoyl ethanolamide source started with concentration of 1.28 ng/ml of each. As can be seen in Fig. 4, the purified rhGM-CSF promoted 50 maximal growth of the TF-1 cells at a concentration of 12.5 pg/ml (4.06109 U/mg) with the commercial rhGM-CSF having comparable activity at 11.4 pg/ml (4.46109 U/mg).Once solubilized, the protein was dialyzed against decreasing amounts of GuHCl. It is common to use dilution to reduce theDiscussionWe had previously developed a protocol that efficiently allowed for the refolding of Fab fragments of antibodies from inclusion bodies [19]. Therefore, because rhGM-CSF forms inclusion bodies in E. coli, we adapted our refolding protocol for its purification. Our protocol is based on a combination of strategies for refolding of inclusion body proteins. For a review of isolation and purification of proteins from inclusion bodies see Vallejo and Rinas [12]. After isolation of the inclusion bodies from the E. coli cell lysate, the first step in the rhGM-CSF refolding procedure is the solubilization of the inclusion bodies. (Our previous work refolding Fab fragments found that extensive washing of the inclusion body pellet did not improve the refolding protein yields, so we 1407003 therefore do not include a wash step.) The conditions we used to resuspend the rhGM-CSF were harsh, however they were effective at solubilizing the protein and reducing non-native, interand intramolecular disulfide bonds. As well, although expensive, we found that use of a high grade of GuHCl was necessary to achieve efficient refolding.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 rhGMCSF 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 6SD of triplicate measurements. doi:10.1371/journal.pone.0049.Ly 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. doi:10.1371/journal.pone.0049891.gPurification of Recombinant Human GM-CSFFigure 3. Mass spectral analysis of purified rhGM-CSF. (A) Shown is the sequence of the expressed rhGM-CSF. In bold and underlined is rhGMCSF sequence coverage obtained by the 18334597 mass spectral analysis. 95 sequence coverage was obtained, not including the 86His 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 colonystimulating factor (amino acid residues 63?7) digested by protease V8, is shown as a representative spectra. M: Methionine oxidation; C: Cystine carbamidomethyl modification. doi:10.1371/journal.pone.0049891.gTools was performed. The titrations of purified rhGM-CSF or the commercial source started with concentration of 1.28 ng/ml of each. As can be seen in Fig. 4, the purified rhGM-CSF promoted 50 maximal growth of the TF-1 cells at a concentration of 12.5 pg/ml (4.06109 U/mg) with the commercial rhGM-CSF having comparable activity at 11.4 pg/ml (4.46109 U/mg).Once solubilized, the protein was dialyzed against decreasing amounts of GuHCl. It is common to use dilution to reduce theDiscussionWe had previously developed a protocol that efficiently allowed for the refolding of Fab fragments of antibodies from inclusion bodies [19]. Therefore, because rhGM-CSF forms inclusion bodies in E. coli, we adapted our refolding protocol for its purification. Our protocol is based on a combination of strategies for refolding of inclusion body proteins. For a review of isolation and purification of proteins from inclusion bodies see Vallejo and Rinas [12]. After isolation of the inclusion bodies from the E. coli cell lysate, the first step in the rhGM-CSF refolding procedure is the solubilization of the inclusion bodies. (Our previous work refolding Fab fragments found that extensive washing of the inclusion body pellet did not improve the refolding protein yields, so we 1407003 therefore do not include a wash step.) The conditions we used to resuspend the rhGM-CSF were harsh, however they were effective at solubilizing the protein and reducing non-native, interand intramolecular disulfide bonds. As well, although expensive, we found that use of a high grade of GuHCl was necessary to achieve efficient refolding.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 rhGMCSF 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 6SD of triplicate measurements. doi:10.1371/journal.pone.0049.