rhG-CSF analyses.

<p><b>A</b>. RP-HPLC chromatogram of rhG-CSF. Samples (30 μg) containing rhG-CSF for analysis was loaded on the RP-HPLC column. <b>B</b>. GPC-HPLC chromatogram of rhG-CSF. <b>C</b>. MALDI mass spectra of rhG-CSF.</p

Cell growth and rhG-CSF expression during the fed-batch culture.

<p><i>E. coli</i> JM109/pPT-G-CSF cells were grown in fed-batch culture using temperature shift method in a 300-L fermentor with glucose as the energy source. Optical density was detected using a spectrophotometer at 600 nm. Glucose concentration (grey triangle); OD₆₀₀ (●); Expression rate of rhG-CSF (□). The arrow indicates the start time of feeding.</p

Biological activity of rhG-CSF protein.

<p>The rhG-CSF protein was assessed for its ability to stimulate the proliferation of an NSF-60 cells. The cells were incubated for 48 h in the presence of rhG-CSF. As a control, standard hG-CSF (WHO 2^nd International Standard) was also analyzed. The bioactivity of rhG-CSF on NFS-60 cell proliferation was measured using the reagent WST-1. Data are the mean ± SD of triplicate measurements (significant versus control, p<0.05).</p

The analysis of rhG-CSF protein by SDS-PAGE and IEF.

<p><b>A</b>. A 4–12% discontinuous NuPAGE SDS-PAGE gel and Coomassie brilliant blue staining were used to confirm the purity of rhG-CSF. Lane 1, molecular weight marker; lane 2, standard rhG-CSF (Filgrastim); lane 3, rhG-CSF. <b>B</b>. The Novex pH 3–10 IEF gel was used to examine the purity of rhG-CSF. The IEF marker indicates pI. Lane 1, pI marker 4.5–7.4; lane 2, standard hG-CSF; lane 3, purified rhG-CSF. The arrow indicates rhG-CSF.</p

Analysis of rhG-CSF protein by SDS-PAGE.

<p>Upon IPTG induction, rhG-CSF was analyzed using a 4–12% reducing SDS-PAGE gel followed by Coomassie brilliant blue staining. <b>A.</b> Lane 1, cell homogenates of <i>E. coli</i> JM109/pPT-G-CSF without IPTG induction; lane 2, cell homogenates after IPTG induction for 1 h; lane 3, After IPTG induction for 3 h; lane 4, After IPTG induction for 5 h. <b>B.</b> Lane 1, total homogenates; lane 2, supernatant after centrifugation; lane 3, Pellet after centrifugation. Most of IPTG induced rhG-CSF is pelleted after centrifugation. The arrow indicates rhG-CSF.</p

Prep-HPLC chromatogram and IEF analysis.

<p>Prep-HPLC chromatography was performed to detect rhG-CSF protein. <b>A</b>. Chromatogram of prep-HPLC with the sup after pH precipitation of refolded rhG-CSF. <b>B</b>. IEF analysis of each fraction from A to H obtained by Prep-HPLC. The IEF marker indicates pI. Lane 1, pI marker 4.5–7.4. Absorbance is in milliabsorbance units (mAU).</p

The purity of rhG-CSF is increased following refolding from inclusion bodies.

<p>Insoluble fraction of the induced culture were harvested, refolded, and precipitated under the stepwise decrease of pH (7.5→5.5). The refolded samples before and after pH precipitation process were analyzed by reducing 4-12% SDS-PAGE, followed by Coomassie brilliant blue staining. Lane 1, solubilized IBs; lane 2, refolded IBs before pH precipitation; lane 3, supernatant (Sup) after pH 7.5 precipitation; lane 4, sup after pH 6.5 precipitation; Lane 5, sup after pH 5.5 precipitation. The arrow indicates rhG-CSF.</p

Peptide map and Western blot analysis of standard hG-CSF and purified rhG-CSF.

<p><b>A</b>. Two chromatograms of standard hG-CSF and rhG-CSF are overlapped for comparison. The solid arrow is the chromatogram for rhG-CSF and the dotted arrow is the chromatogram for standard hG-CSF. Absorbance is in absorbance units (AU). <b>B</b>. Two rhG-CSF proteins were examined by western blot after a 4–12% reducing SDS-PAGE performance. Lane 1, standard rhG-CSF (Filgrastim, 5 μg); lane 2, purified rhG-CSF (5 μg). The arrow indicates rhG-CSF.</p