A three-level four-factor Behnken experimental design was applied to statistically investigate the effects of four independent cultivation variables on the soluble expression of anti-HER2 immunotoxin in E. coli BL21(DE3). The input factors include IPTG concentration (at three levels: 0.1, 0.55 and 1 mM), post-induction time (at three levels: 2, 10 and 18 h), post-induction temperature (at three levels: 23–37 ºC), and medium recipe (at three categories: LB, 2XYT, and TB), whereas the quantitative factor levels were coded as − 1 (low level), 0 (central point level), and 1 (high level) (Table 2). As a result, 45 cultivation runs with three replications (i.e., runs 4, 7, and 19 (for LB), 11, 13, and 28 (for 2xYT), and 3, 29, and 42 (for TB)) at center point were conducted in parallel (Table 3). For LB medium, runs 4, 5, 6, 7, 14,18, 19, 21, 25, 30, 33, 34, 35, 39, and 43 were performed simultaneously from one seed culture (i.e., overnight culture in LB). The same seed culture (i.e., overnight culture in TB) was used to prepare samples of runs 3, 8, 9, 12, 16, 22, 27, 29, 31, 36, 37, 41, 42, 44, and 45 for TB medium. For 2xYT medium, runs 1, 2, 10, 11, 13, 15, 17, 20, 23, 24, 26, 28, 32, 38, and 40 were performed simultaneously from one seed culture (i.e., overnight culture in 2xYT).

Data analysis of the experimental design was carried out using the Design Expert software (version 13.0.5.0, StatEase Inc., Minneapolis, USA) to develop a model. The good fit of the model was confirmed by measuring the difference between actual and predicted values for each point (i.e. studentized residuals).

After protein expression for each cultivation, bacterial cells were collected by centrifugation at 3,500 g for 20 min, and the resulting cell pellet was resuspended in phosphate-buffered saline (PBS). For cell disruption, the suspension was sonicated for five cycles of 1 min with an interval of 1 min on ice between cycles. The soluble and insoluble fractions of the cell were separated using centrifugation at 7,500 g and 4 °C for 15 min.

To evaluate the expression level and solubility of the expressed anti-HER2 immunotoxin, the soluble fraction of the sonicated culture sample, insoluble and total protein fraction were analyzed using a 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The intensity of the corresponding bands, soluble and total proteins, were assessed by Gel Analyzer 19.1 software (Lazar Software, Hungary) and the amount of the target protein was estimated based on the standard protein (i.e., bovine serum albumin) (Table 3). Then, the percentage ratio of the soluble and the insoluble protein to the total protein was determined (Table 2).

The soluble fraction of the culture sample was used to purify anti-HER2 immunotoxin using affinity chromatography under native conditions, as described in our previous study with some modifications (Akbari et al. 2015a). The protein concentration in each purification fraction was determined using the Bradford method (Malekian et al. 2019).

To evaluate the bioactivity of the purified anti-HER2 immunotoxin, two cancer cell lines, i.e., the HER2-overexpressing cell line of SK-OV-3 and the HER2-low-expressing cell of MCF-7, were seeded in a 96-well ELISA plate at a cell density of 2 × 105 cells/ml and 37 ºC overnight. The cells were rinsed with PBS and then immobilized with 4% formalin buffer for 20 min. After that, the plate was washed thrice with PBS and then treated with 1% bovine serum albumin in PBS for 2 h at the room temperature. Then, samples containing anti-HER2 immunotoxin in a serial dilution were added to individual wells and incubated for 2 h at 25 ºC with gentle shaking. Four wells were only treated with PBS (i.e., no immunotoxin was added) as negative control. Each well was rinsed for three times with 0.05% Tween 20 in PBS. HRP-conjugated anti-His antibody (1: 50,000 dilution) was added to each well for incubation for 2 h at the room temperature with gentle shaking. After washing three times, OPD substrate was added to each well for incubation for 20 min. The colorimetric reaction was terminated by addition of 10 µl of 2M H2SO4 to each well, and the absorbance was measured at 450 and 490 nm (Akbari et al. 2014).