Human cell lines and serums
Human normal (HEK293), AML (KG-1and HL-60), and CML (K-562) cell lines were purchased from Iran National Cell Bank (INCB, Iran). HEK and KG-1 were chosen for no or low expression of IL-1RAP and the two other lines were selected for overexpression of IL-1RAP. Cells were cultured and maintained in Dulbecco’s Modified Eagle’s Minimal essential medium (DMEM) (for HEK) or Roswell Park Memorial Institute (RPMI) 1640 medium (for KG-1, HL-60, and K-562) plus Glutamine (BioIdea, Iran), 10% v/v fetal bovine serum (FBS) (Gibco, USA) and 1% Penicillin/Streptomycin (100 U/mL and 100 µg/mL, respectively) in a humidified incubator, at 37 ˚C with 5% CO2.
Normal serum was voluntarily obtained from one of the authors and leukemic serum from a 59 years old male patient with relapsed AML and high leukemic burden before treatment initiation.
Sequences, constructions, and bioinformatics
Sequence extraction, design, gene synthesis, and cloning procedures
The E. coli codon-optimized sequence of the anti-IL-1RAP scFv (fused to fusion format of streptavidin, GenBank accession number: OM642337) in pET28a (Fig. S1a) was already present in our lab. The sequence of the scFv had been formerly extracted from a patent (PCT/US2013/077323 [15]: SEQ ID NO 13 for VH and SEQ ID NO 14 for VL) (Fig. S1b). The scFv was constructed as VH-(Gly4Ser)5-VL-Gly4-6xHis (Fig. S1c) by removal of downstream sequences and substitution with a Gly4 coding oligo (Fig. S1d).
Secondary structure prediction
We utilized two webservers, 2Struc [16] and PSIPRED [17] for secondary structure predictions. The scFv constructs were modeled using I-TASSER [18]. Model 1 from each protein was employed for analysis by 2Struc. For PSIPRED, the amino acid (AA) sequences were submitted.
Exploration of aggregation hotspots
Potential aggregation regions were investigated in silico by five different webservers (inspired from [19]). For AGGRESCAN [20], Tango [21], Waltz [22], and FoldAmyloid [23], the AA sequences of the scFv-6xHis in FASTA format were inputted. For the AGGRESCAN3D (A3D) predictor [24], I-TASSER generated 3D model of the molecule was uploaded as an input. All predictions were accomplished with the default input parameters settings of tools.
Preliminary expression of periplasmic and cytoplasmic constructs
The periplasmic and cytoplasmic constructs were transformed to BL21 (DE3), and transformants were cultured overnight (ON) in a 5 ml Luria-Bertani (LB) medium at 37 ˚C. The ON cultures were refreshed and induced at Abs600nm of ~0.5 after obtaining before induction (BI) samples. pET26b(+)/BL21 (DE3) culture was induced with 0.1 mM isopropyl-b-D thiogalactopyranoside (IPTG) and continued ON at below 30 ˚C, 150 revolutions per minute (rpm), while pET28a/BL21 (DE3) culture was induced with 0.5 mM IPTG and continued for 4 h at 37 ˚C, 150 rpm.
Analysis of expression samples
For analysis of expression in total cell fractions (TCFs), cellular pellets of before and after induction (AI) samples were resuspended in dH2O. To study the soluble and insoluble fractions, the pellets of un-induced and induced (4 h, ON) cultures from three different temperature conditions were resuspended in phosphate-buffered saline (PBS) and lysed with sonication. After centrifugation (⁓11,000 g for 10 min), supernatants were collected as soluble fractions. The sediments were washed with PBS and then dissolved in 0.5 ml of 8 M urea and then centrifuged. The supernatants of the second step were collected as insoluble fractions. All samples were mixed with an appropriate volume of Laemmli buffer (with 2% 2-mercaptoethanol (2ME)) and boiled for ⁓10 min before sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Western Blot (WB) Analysis
WB on expressed scFv
Samples were subjected to SDS-PAGE using 12% polyacrylamide gel. After electrophoresis, protein bands were transferred to the nitrocellulose membrane by the semi-dry method. The membrane was blocked (3% w/v skim milk in PBS) ON at 4 ˚C and after 4 times washing the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-His-tag Ab (Sigma-Aldrich, USA). The membrane was then washed five times, and His-tagged scFv bands were developed by 3, 3’-Diaminobenzidine (DAB) substrate solution.
WB on lysates of leukemic cells
K-562 and KG-1 cells were harvested (400g, 5 min) and lysed by resuspension in minimum volumes of lysis buffer (Table S2). Total protein contents were estimated using Bradford assay, and equal protein quantities from each sample were loaded on 12 % gel and subjected to SDS-PAGE. The protein bands were transferred to the nitrocellulose membrane. The membrane was blocked, washed, and then incubated with the anti-IL-RAP scFv solution (⁓2.5 µg scFv per 1ml PBS). The membrane was then washed and incubated with HRP-conjugated anti-His-tag Ab, and the experiment was continued as mentioned above.
Isolation of IBs for refolding
A single colony of transformant was cultured and induced with 0.2 mM IPTG at 37 ˚C, ON. After harvesting the culture (⁓4000 g, 15 min) the cellular pellet was resuspended in 2 ml lysis/wash buffer (Table S2) plus ⁓5 mg lysozyme and incubated for about 1 h, at RT with occasional shaking. The suspension was volumized to 30 ml with the same buffer and lysed using two rounds of sonication (10 s pulses/20 s rest intervals for 20 times at 40% amplitude). IBs were separated by centrifugation, resuspended in the buffer mentioned above, and, then sonicated. Washed IBs were isolated using centrifugation and then washed with PBS to remove residual ethylenediaminetetraacetic acid (EDTA) and detergent. High-quality IBs were stored at -20 C until solubilization.
Refolding and purification of the scFv
We compared three different methods for refolding scFv from IBs produced in BL21 (DE3). In all, 2.5 mg denatured IB (denatured in solubilization buffer, Table 1) with ≥75% purity was introduced, and all the procedures were done in the same buffer condition (50 mM Tris-HCl, 150 mM NaCl, pH 8). Denatured protein samples in all practices were prepared from the stock solution (10 mg/ml protein in solubilization buffer plus 10 mM 2ME). We used 2 mM cysteine and 0.4 mM cystine in all methods but at different urea concentration points. After refolding and before purification, aggregates were removed using centrifugation (⁓10,000 g for 30 min). The refolded protein samples were buffer exchanged to PBS and concentrated using a 10 kDa cut-off AmiconTM Ultra-0.5 Centrifugal Filter (Merck). The final concentrations were determined using a UV spectrophotometer (Abs280nm at the related extinction coefficient (Table S3)). The recovery rates were calculated using the formula [1].
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)
Table 1. The buffer components used in refolding procedures (inspired by [9] and [13])
pH
|
2ME
|
Imidazole
|
L-cysteine
|
L-cystine
|
Urea
|
L-Arg
|
NaCl
|
Tris-HCl
|
Components*
|
8
|
-
|
25
|
-
|
-
|
8000
|
-
|
150
|
50
|
Solubilization buffer
|
8
|
1
|
25
|
2
|
0.4
|
500
|
500
|
150
|
50
|
Refolding buffer A
|
8
|
1
|
25
|
0 or 2
|
0 or 0.4
|
-
|
-
|
150
|
50
|
Refolding buffer B
|
*Concentrations in mM
Refolding by stepwise dialysis
In refolding by dialysis, 0.25 ml of stock solution (2.5 mg protein) was added to the 24.75 ml solubilization buffer. The resulting solution (25 ml of 100 µg/ml) was transferred into the dialysis bag with a 12 kDa cut-off. The bag was immersed in the same buffer (but with 6 M urea). The urea concentration decreased step by step (4 M, 2M, 1M, and 0.5 M). The refolding buffers from 2M onward contained 1 mM 2ME, 2 mM L-cysteine, and 0.4 mM L-cystine. The buffer was then exchanged to refolding buffer B, and dialysis continued ON. The protein solution was brought out of the bag after final dialysis in binding/wash buffer (Table S2) and then applied on Ni-NTA resin (QIAGEN, Germany) pre-equilibrated with binding/wash buffer. After complete washing with the same buffer, the refolded proteins were eluted from the resin using the elution buffer (Table S2).
On-column refolding and purification of solubilized IBs
For on-column refolding, 0.25 ml of stock solution (2.5 mg protein) was added to a 9.75 ml solubilization buffer. The denatured protein solution (10 ml of 250 µg/ml) was loaded on the Ni-NTA column pre-equilibrated with the same buffer. The column was thoroughly washed with solubilization buffer (plus 1 mM 2ME), and then a gradient of refolding buffer B (Table 1) was applied from 0% to 100%. Inspired by a previous report [25] since the 3M urea point, the refolding buffer B contained 2 mM L-cysteine and 0.4 mM L-cystine. The column was then thoroughly washed with refolding buffer B, and the refolded proteins were eluted from the resin as mentioned above.
Refolding using pulse dilution method
For the dilution method, 0.25 ml of stock solution (2.5 mg protein) was added to the 0.75 ml solubilization buffer. In the first step, the resulting protein solution (1 ml of 2.5 mg/ml) was diluted drop-by-drop into 24 ml of cold refolding buffer A (Table 1) without stirring. The final protein concentration, if no aggregation occurred, would be 100 µg/ml. After ˃24 h incubation at 4 ˚C, the protein solution was dialyzed (at 4 ˚C, ON) in refolding buffer B (Table 1) without shuffling pairs and then dialyzed in binding/wash buffer (Table S2) before loading on Ni-NTA resin. The purification process was continued as mentioned above.
Cell-based ELISA
All solutions were prepared with Hank’s balanced salt solution (HBSS) (Table S2), and the plate was kept cold throughout the process. At first, the wells were pre-coated with high molecular weight (HMW) polyethyleneimine (50-100 kDa PEI, MP Biomedicals (FKN ICN BIOMED) the USA, CAS number: 9002-98-6) 100 µl of PEI solution (0.1 mg/ml in dH2O) was added to the wells. The plate was incubated at 37 ˚C to be completely dried. After pre-coating, 100 µl of washed cells were added to the wells (≥5×104/well) and allowed (4 h) to be settled. The cells were then fixed by slow addition of 0.5% v/v glutaraldehyde solution (100 µl/well). After 1 h, the wells were washed and blocked with 1% w/v bovine serum albumin (BSA) (200 µl/well) for 4 h. After washing, 50 µl of the produced scFvs (50 µg/ml) were added to the wells. The plate was incubated at 4 ˚C, ON, and after 4 times washing, 100 µl of mouse HRP-conjugated anti-His-tag Ab (1:1000) was added to the wells. Ab solution was removed and wells thoroughly washed before adding 3,3’,5,5’-tetramethylbenzidine (TMB) substrate solution. The reactions were stopped with sulfuric acid, and the plate was read at 450 nm wavelength.
Proliferation inhibition assay
AML (KG-1 and HL-60), CML (K-562), and normal (HEK293T) human cell lines were seeded on a 96-well plate (2×104/well) in triplicate. To wells 100 µl of serially diluted antibody samples (5000 to 8 ng/µl) were added and after 48 h incubation (37 ˚C), 20 µl of alamarBlueTM reagent was added to the wells. After 3 h additional incubation at 37 ˚C, the plate was read at 570 and 600 nm, and the % Reduction of alamarBlue Reagent for each readout was calculated according to the instruction [26].
Size exclusion chromatography
In order to analyze the homogeneity of dilution-refolded scFv preparation, size exclusion chromatography (SEC) was carried out according to the following method. 100 µL of purified Ab (0.37 μg/μl) was injected into a TSK-gel G2000 SWXL column (7.8 × 300 mm, Tosoh Bioscience, Tokyo, Japan) connected to a Shimadzu HPLC system (Kyoto, Japan). The sample was eluted with an isocratic mobile phase (Table S2) with a 0.5 ml/min flow rate (25 ºC).
Analysis of secondary structure by CD
The secondary structures of three Ab preparations were figured out by far-UV CD spectroscopy (Jasco J-810 Spectropolarimeter, Japan). The ellipticity (θ) of samples was measured at 190 to 240 nm wavelength. The samples’ buffer was extensively exchanged to dH2O using a 10 kDa cut-off AmiconTM Ultra-0.5 Centrifugal Filter prior to the measurements. The analyses were performed at RT by injection of ⁓300 µl of each sample into a 1 mm quartz cuvette.
Statistical analysis
The statistical analysis of data was accomplished using GraphPad Prism version 8.0 for Windows (GraphPad Software, La Jolla, CA, USA). The significance level between the groups was analyzed using one-way ANOVA.