INNO The design and analysis of chimeric protein properties of 390 residues from N-terminal of DT (lacking 21 amino acids of signal sequence from the N-terminal of the protein) was selected from online gene banks and fused to the amino acid sequence of STxB. Several hydrophobic linkers were tested to choose the top linker which sustains functionality of the standard structure of the two parts of the recombinant protein. Finally, GGGGSGGGGSGGGGS sequence was designated as the linker to maintain the flexibility of the construct (Figure 1A).
Afterwards, the amino acid sequence was back-translated and nucleic acid codons were adjusted based on the codon, considering E. coli as the expression host. The chimeric gene exposed a codon adaptation index (CAI) of 0.62 in comparison to the wild type gene that was 50.15%. The GC% codon usage bias in E. coli was augmented by upgrading GC% and CAI to 50.15% (GC% of E. coli is nearby 50) and 0.62, respectively. The CAI of >0.8 was considered statistically significant in terms of high gene expression level (Figure 1B).
Furthermore, the essential restriction enzyme sites (EcoRI and HindIII) were introduced at the ends of the sequence for cloning purposes.

The mRNA Structure Prediction
The secondary structure of the dt390-stxB mRNA was predicted using the online software mfold. To specify the anticipated folding of the designed gene, a genetic algorithm-based RNA secondary structure prediction was associated with comparative sequence analysis. The folding of 5' terminal region of the gene had the same structure as the bacterial gene. The minimum free energy (MFE) was estimated for RNA secondary structure; all 29 structural elements recovered in this study had the same the folding as RNA constructs. The obtained data demonstrated that the mRNA had the sufficient stability for the effective translation in the novel host.
 Results are demonstrated in Figure 2 and Table 1. The free energy of thermodynamic cooperative units associated with this structure was ΔG=-427.40 kJ/mol.
Random coils established to be recurrent 189, 81 extended strand and 189 alpha helix (Table 2 and Figure 1C).

Chimeric Protein Properties
ProtParam web server was used to reveal the physiochemical properties of the DT390-STxB sequence. The number of amino acids was found to be 443, the average molecular weight was 53 kDa, and the pI was 5.11. In addition, the total number of positively charged residues (Arg+Lys) was 42 and total number of negatively charged residues (Asp+Glu) was 56. The instability index of DT390-STxB was computed by the ProtParam web server, which appeared to be 36.95 (Table 3, 4).

Table 1. Thermodynamic details related to 5’ end of chimeric construct DT390-STxB mRNA. According to the minimum free energy (ΔG) of the 5' end of chimeric mRNA, their initial ATG is constrained in hairpin loop structure.

Table 2. Arrangement of secondary structure of origin and chimeric protein

Table 3.Parameters calculated by ExPSy’s ProtParam tool

Cancer is the most common cause of death in many developed countries. Today, the targeted therapies like the use of immunotoxins are increased which target specific antigens or receptors on the surface of tumor cells. Immunotoxins are recombinant proteins, which consist of a tumor-specific ligand-like antibody and a toxic protein. Ligand-targeted therapy makes it possible for tumor specificity and limited toxicity and promises for developing novel therapies for cancer treatment. It can carry higher doses of a drug to the tumor tissue and may overcome obstacles presented by cytotoxic chemotherapy (33-40).
Native DT is a 538-amino acid protein consisting of three domains. Part A is the enzymatically active domain, part B has a hydrophobic domain at the N-terminal portion, and the C-terminal portion of fragment B is the receptor-binding domain. In this study, we evaluated DT390-STxB chimeric protein as a new anti-tumor candidate via genetically fusing the DT390 fragment of DT538 to STxB (41-43).
In a study carried out by Imani-Fooladi et al., the genetically fused protein called the TGFαL3-SEB fusion protein was designed and evaluated as a novel anti-tumor candidate. This protein was constructed by fusing the third loop of transforming growth factor alpha (TGFαL3) to the staphylococcal enterotoxin type B. The CAI index of the TGFαL3-SEB fusion protein enhanced from 0.5 in the wild type to 0.85 in the chimeric optimized gene. Moreover, the overall guanine-cytosine (GC) content decreased from 45.83% to 44.06%, hence increasing the overall stability of mRNA of the synthetic gene. The random coil was the second most available structure in this experiment. The pI of the hydrophilic TGFαL3-SEB fusion protein was 7.72. The highest C-score of -0.42 was annotated for tertiary structure of the TGFαL3-SEB protein (44). The comparison between the two chimeric proteins showed that the TGFαL3-SEB and DT390-STxB constructs are stable enough and have sufficient affinity to the overexpressed cancer cell receptors.
In another study by Keshtvarz et al., PE38-P4A8 chimeric immunotoxin was designed and evaluated. The GC content and codon bias of both wild and synthetic form of the protein were assessed and the results for optimized gene showed no rarely used codon, and the codon bias was demonstrated in E. coli as the bacterial host. The optimized GC content was about 54.2% and the CAI value was 0.94 revealing the high and stable expression in bacterial cells. The highest C-score of -3.36 observed in tertiary structure of the fusion protein. For isolating two parts of the proteins, the ASGGPE and (G4S)₃ linkers were recruited (45).
 In silico studies are able to complete trans-criptional and translational gene fusion, other than the quality expression of the suggested concept in host expression vectors. CAI is the main factor used for gene optimization within a range of 0-1 and an ideal value of 1.0. Subsequently, our objective was to construct a fusion protein that is expressed in E. coli as a host expression vector. So, the codon table of E. coli was selected for back-translation of the amino acid sequence and ideal expression of the construct. In our gene, CAI index was improved from 0.6 in the wild type sequences to 0.9 in the chimeric optimized gene. Furthermore, the overall GC content was reduced from 50.03% to 47.58%, which should increase the overall stability of mRNA from the synthetic gene. Codon optimization contributes to improve the expression of the synthetic construct in the desired host vector.
The mRNA structure optimization was established based on the low ΔG value and the MFE values of the start codon. These characteristics can support ribosome binding and translation initiation. For prediction of RNA secondary structure, a genetic algorithm-based approach along with relative sequence analysis was recruited to define the prospective folding of the chimeric gene. The 5' terminus of the gene was folded in a way which is typical for all bacterial gene structures. The MFE for secondary structures formed by RNA molecules was also predicted. The mRNA secondary structure of the chimeric gene was analyzed using the mfold program with the parameters as follows: linear RNA folding at 5%, window=20, and max folds=50. All 32 structures achieved in this analysis exposed folding of the RNA construct at 37°C with initial ΔG ranging from -543.80 to -481.78 kcal/mol. The best structure (ΔG=-427.40 kJ/mol) is shown in Figure 4. The data showed that the mRNA was stable enough for proper translation in the new host.
ProtParam was used to determine the physio-chemical properties of the protein sequence. The results of the primary structure analysis showed that DT390-STxB fusion protein is hydrophilic in nature due to the large number of polar residues. Also, the primary structure analysis suggests that the average molecular weight of DT390-STxB is considered to be 53.64 kDa. The pI of a protein is defined as the pH at which the net charge on the surface of the protein is zero. At pI, the proteins are stable and dense. The calculated pI value of DT390-STxB is 5.11 (pI<7). Although ExPASy’s ProtParam computes the extinction coefficient (ɛ) for a range of  wavelengths (276, 278, 279, 280, and 282 nm), extinction constant (K) of DT390-STxB at 280 nm is 49850 M^–1 cm^–1 with respect to the low concentration of Cys, Trp, and Tyr, which indicates that this fusion protein cannot be analyzed using UV spectral methods.
The three-dimensional (3D) structure of the proteins is of a major importance to provide insights into their molecular functions. The 3D model of the recombinant DT390-STxB protein was produced using the I-TASSER online server, which generates 3D models along with their C-score, Z-score, RMSD, and TM-score. This server was generated in three models with C-scores as follows: -2.82, -3.32, and 0.03. Among the three models, model one was selected for further analysis as it assigned the highest C-score (0.03). The expected TM-score was 0.72±0.11, which approved the validity of the model. A TM-score more than 0.5 probabilities accuracy of topology. Subsequently, by generating 3D model, structural assessment and stereo chemical analyses were accomplished using Procheck Ramachandran plot. Energy minimization was determined by analysis of 3D structural stability of the chimeric protein using Swiss-PdbViewer. The proportion of residues was 84.5% in the favored region, 9.7% in allowed region, and 5.8% in the outlier region. Also, the assessment of model stability of the Ramachandran plot indicated that the most residues of the chimeric model are in a stable zone. The model was analyzed by different DT390-STxB fusion proteins as a new anti-tumor candidate. Since it is important to establish the structure-function relation of DT390-STxB fusion protein, some experimental studies have already started investigating the DT390-STxB fusion protein by several tools and software.
In this study, we introduce a novel anti-tumor fussion protein. Our bioinformatics results show that this protein was a stable chimeric protein and it can be used for treatment against breast cancer.