Soluble Cytoplasmic Expression and Purification of Immunotoxin HER2(scFv)-PE24B as a Maltose Binding Protein Fusion

Abstract

Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC₅₀ values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.

Keywords: HER2(scFv)-PE24B; Pseudomonas aeruginosa; Pseudomonas exotoxin A; cytotoxicity; immunotoxin; maltose binding protein; protein expression; protein purification; trastuzumab.

Figure 1

Design and construction of the HER2(scFv)-PE24B immunotoxin fusion protein. (A) Schematic representation of MBP-HER2(scFv)-PE24B. (B) The MBP-HER2(scFv)-PE24B plasmid was constructed by the multisite Gateway cloning method.

Figure 2

Expression and solubility of MBP-HER2(scFv)-PE24B in different E. coli hosts. Expression of the MBP fusion protein was induced by 0.5 mM IPTG at 37 °C (A) and 18 °C (B). The arrows indicate MBP-HER2(scFv)-PE24B (97.4 kDa). M, molecular weight size markers; C, total proteins of BL21(DE3) before IPTG induction (negative control); I, total proteins after IPTG induction; P, insoluble pellet fraction after cell sonication; S, soluble fraction after cell sonication. (C) Expression levels and (D) solubility levels at 37 °C and 18 °C for the three E. coli hosts. The expression and solubility levels were analyzed using the densitometry method and three replicate experiments. The expression levels (%) of MBP-HER2(scFv)-PE24B were calculated based on the density ratio of MBP-HER2(scFv)-PE24B to total E. coli proteins. Solubility levels (%) were calculated based on the density ratio of solubly expressed MBP-HER2(scFv)-PE24B to the total expressed MBP-HER2(scFv)-PE24B.

Figure 3

Purification of HER2(scFv)-PE24B in BL21(DE3). (A) Flow chart of the purification steps. IEC, ion exchange chromatograph; TEV, tobacco etch virus; IMAC, immobilized metal affinity chromatography; GPC, gel permeation chromatography. (B) MBP fusion-derived HER2(scFv)-PE24B purified from BL21(DE3) M, molecular weight size markers; lane 1, total cell proteins before IPTG induction (negative control); lane 2, soluble proteins after cell sonication from total cell proteins induced by IPTG; lane 3, MBP-HER2(scFv)-PE24B fusion protein (97.4 kDa) purified by cation exchange chromatography; lane 4, MBP tag cleavage with TEV protease (28.6 kDa) showing the MBP tag (43.9 kDa) and HER2(scFv)-PE24B (53.5 kDa); lane 5, IMAC purification of HER2(scFv)-PE24B after TEV cleavage; lane 6, HER2(scFv)-PE24B (53.5 kDa) purified by gel filtration chromatography.

Figure 4

Determination of the purity of HER2(scFv)-PE24B. (A) Purified HER2(scFv)-PE24B was analyzed by HPLC using a Protein-pak 300SW SEC column to evaluate the purity. The x-axis shows the retention time (min) and the y-axis indicates the absorbance at 280 nm (arbitrary units, AU). The main peak of HER2(scFv)-PE24B is visible at 12.239 min. (B) Fractions from (A) were analyzed using SDS-PAGE. (C) Silver staining of the SDS-PAGE gel to assess the purity of the recombinant immunotoxin HER2(scFv)-PE24B (53.5 kDa).

Figure 5

LC-MS/MS analysis for verification of HER2(scFv)-PE24B. HER2(scFv)-PE24B was digested by trypsin before mass analysis. The percentage coverage for peptide identification was 77.2%. The identified fragments are colored green.

Figure 6

HER2 levels on the surface of four breast cancer cell lines. (A) Purified HER2(scFv)-GFP (54.4 kDa) from E. coli was used for FACS analysis. (B) SKBR3, BT-474, MDA-MB-231, and MCF7 cells were incubated with HER2(scFv)-GFP and stained with DAPI. Red represents cells not treated with HER2(scFv)-GFP and blue indicates cells treated with HER2(scFv)-GFP.

Figure 7

Cytotoxicity of HER2(scFv)-PE24B, HER2(scFv), and PE24B against high HER2-expressing and low HER2-expressing cell lines. HER2(scFv)-PE24B, HER2(scFv), and PE24B were added to high HER2-expressing cell lines SKBR3 (A) and BT-474 (B), and low HER2-expressing cell lines MCF7 (C) and MDA-MB-231 (D) for 72 h. For all cell lines, HER2(scFv)-PE24B showed stronger cytotoxicity than HER2(scFv) or PE24B alone. Untreated cells served as controls. Cell viability was determined from at least three independent MTT assay experiments and calculated as the absorbance ratio of treatment vs. control groups. Data are presented as mean ± standard error (SE).

Figure 8

Correlation of the cytotoxicity of HER2(scFv)-PE24B and the HER2-positive percentage of cell lines. (A) The cytotoxicity of HER2(scFv)-PE24B against each of the four cell lines plotted together. Data are presented as mean ± standard error (SE). (B) IC₅₀ values for HER2(scFv)-PE24B and HER2 expression show a strong negative correlation. As the percentage of HER2-positive cells increases, the IC₅₀ of HER2(scFv)-PE24B decreases. The data were fitted using the power function: IC₅₀ = 2561.2 × (percentage of HER2-positive cells)^−1.023 with an R² value of 0.9917. The key shows the symbols corresponding to MDA-MB-231, MCF7, SKBR3, and BT-474.