Purification of hGCSF from the MBP-hGCSF fusion protein expressed in <i>E. coli</i> BL21(DE3).

<p>A. Overview of the purification process using an MBP affinity column, TEV protease digestion, and IMAC. B. SDS-PAGE analysis of hGCSF at various stages of the purification process. M, molecular weight marker; lane 1, total protein before IPTG induction; lane 2, total protein after IPTG induction; lane 3, soluble fraction after cell sonication; lane 4, MBP-hGCSF fusion protein purified using an MBP affinity column (63.3 kDa); lane 5, MBP-hGCSF fusion protein after cleavage with TEV protease showing separated MBP (40.3 kDa) and hGCSF (18.8 kDa); lane 6, purified hGCSF after IMAC (18.8 kDa). C. IMAC chromatogram of the TEV protease cleaved MBP-hGCSF fusion protein showing clear separation of the MBP tag and hGCSF. D. Silver staining of 7.5 µg of purified hGCSF. M, molecular weight marker; R, reduced hGCSF; NR, non-reduced hGCSF.</p

Cell proliferation assay of purified hGCSF using the M-NFS-60 cell line.

<p>Light microscopy images of M-NFS-60 cells incubated without (A) or with (B–D) 1 ng/mL hGCSF for 48 h. Commercially available hGCSF (B), hGCSF purified from MBP-hGCSF (C), and GCSF purified from PDIb'a'-hGCSF (D) were used. The scale bar represents 100 µm. E. Dose-response curve of M-NFS-60 cells following exposure to different concentrations of purified hGCSF and commercial hGCSF. The number of cells was measured as the OD₅₇₀. •, commercially available hGCSF; ▵, hGCSF purified from MBP-hGCSF; hGCSF purified from PDIb'a'-hGCSF. Data are represented as the mean ± SE of n≥3 of 2 independent experiments. Statistical significance compared to no hGCSF treatment group: *, p<0.05; **, p<0.01; ***, p<0.001.</p

Construction of the hGCSF expression vectors and schematic representations of the domain structures.

<p>A. The method of construction and vector map of the tag-hGCSF construct. All fusion constructs were generated in the same way via LR recombination cloning. Expression of the fusion proteins in <i>E. coli</i> was controlled by the IPTG-inducible T7 promoter, and ampicillin was used as the selection marker. B. Schematic representation of the seven hGCSF fusion proteins used in this study (His6-, Trx-, GST-, PDIb'a'-, MBP-, PDI-, and NusA-hGCSF). The arrow indicates the TEV protease cleavage site. Black is extra sequences from BP and LR recombinations. The amino acid sequence of mature hGCSF is also shown.</p

Expression levels and solubilities of hGCSF fused with seven different N-terminal tags.

<p>Expression levels and solubilities of hGCSF fused with seven different N-terminal tags.</p

The characteristics of hGCSF purified from PDIb'a'-hGCSF and MBP-hGCSF fusion proteins expressed in <i>E. coli</i>.

<p>The characteristics of hGCSF purified from PDIb'a'-hGCSF and MBP-hGCSF fusion proteins expressed in <i>E. coli</i>.</p

Cell proliferation assay of purified hGH in the Nb2-11 cell line.

<p>Dose-response proliferation curve of Nb2-11 cells by exposure to different concentrations of purified hGH and commercial hGH.•, commercial hGH; △, hGH from Trx-hGH; ◊, hGH from Trx-hGH in presence of DTT; □, hGH from MBP-hGH.</p

Expression level and solubility of hGH with seven different tags.

<p>Expression level and solubility of hGH with seven different tags.</p

Schematic representation of domain structure and generation of the MBP-hGH construct.

<p>(A) Vector map of pHMGWA-hGH using the gateway cloning method. Expression of the fusion proteins in <i>E. coli</i> is controlled by the IPTG-inducible T7 promoter, with ampicillin as the selection marker. (B) Schematic structure of the seven fusion proteins His6-, Trx-, GST-, MBP-, NusA-, PDIb′a′-, and PDI-GH (total size). The arrows indicate the TEV protease cleavage site.</p

MALDI-TOF MS analysis of purified hGH from Trx-hGH in <i>E. coli</i> (no DTT during purification).

<p>(A) Tryptic peptide map of hGH (191 aa). (B) MALDI-TOF MS for purified hGH in reducing conditions. (C) MALDI-TOF MS for the purified hGH in non-reducing conditions. MALDI-TOF MS analysis of reduced and non-reduced hGH obtained from MBP-hGH and PDIb′a′-hGH showed similar results (data not shown).</p

hGH purification from Trx-hGH expressed in <i>E. coli</i>.

<p>(A) Flowchart of the purification. (B) Trx-hGH was purified from <i>E. coli</i> with a combination of IMAC and gel filtration chromatography. M, molecular weight marker; lane 1, total cell protein before IPTG induction as a negative control; lane 2, total cell protein treated with IPTG; lane 3, soluble fraction after cell sonication; lane 4, Trx-hGH fusion protein purified using IMAC (37.4 kDa); lane 5, Trx tag cleavage with TEV protease: Trx (15.4 kDa) and hGH (22 kDa); lane 6, final purified hGH (22 kDa). Lane 5 shows that the Trx tag was almost completely cleaved. (C) Gel filtration chromatogram of PDIb′a′-hGH after second IMAC. hGH and oligomers were separated by their sizes. (D) Purity of final product hGH was evaluated by silver staining. M, molecular weight marker; hGH: final product in non-reducing conditions.</p