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

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

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

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

Size Regulation and Stability Enhancement of Pt Nanoparticle Catalyst via Polypyrrole Functionalization of Carbon-Nanotube-Supported Pt Tetranuclear Complex

A novel multiwall carbon nanotube (MWCNT) and polypyrrole (PPy) composite was found to be useful for preparing durable Pt nanoparticle catalysts of highly regulated sizes. A new pyrene-functionalized Pt₄ complex was attached to the MWCNT surface which was functionalized with PPy matrix to yield Pt₄ complex/PPy/MWCNT composites without decomposition of the Pt₄ complex units. The attached Pt₄ complexes in the composite were transformed into Pt⁰ nanoparticles with sizes of 1.0–1.3 nm at a Pt loading range of 2 to 4 wt %. The Pt nanoparticles in the composites were found to be active and durable catalysts for the <i>N</i>-alkylation of aniline with benzyl alcohol. In particular, the Pt nanoparticles with PPy matrix exhibited high catalyst durability in up to four repetitions of the catalyst recycling experiment compared with nonsize-regulated Pt nanoparticles prepared without PPy matrix. These results demonstrate that the PPy matrix act to regulate the size of Pt nanoparticles, and the PPy matrix also offers stability for repeated usage for Pt nanoparticle catalysis