Introduction
Elastin-Like Polypeptides (ELPs) are a group of temperature-sensitive biopolymers with various biotechnological and biomedical applications [6]. ELP is a component of temperature-sensitive synthetic biopolymers, with its solubility dependent on temperature change; under the Transition temperature (Tt), ELP is soluble in aqueous medium and accumulates in the form of self-assembly with increased solution temperature. By binding the protein at the gene level to the ELP, the resulting aggregated proteins will have the same properties [7]. Due to the thermal sensitivity of the ELP molecule, purification by chromatography is not required for protein purification [5]. Studies to produce the fusion of recombinant protein bound to the ELP tag, as well as the oligomerization of ELP, require several cloning steps in the nonspecific vector and re-subcloning [13, 15]. Therefore, in this study, we optimized the pET expression vector, the pET-MOD specific vector, to oligomerize ELP, as well as to produce recombinant ELP tag fusion protein for biotechnological and pharmaceutical applications. Accordingly, the inexpensive purification of the recombinant protein by the Inverse Transition Cycling (ITC) technique and the targeted drug conjugate delivery, etc. will be generated.
Materials and methods
The MOD gene was designed with the structure required for the cloning, expression, and purification of the recombinant protein. Then, the XbaI and XhoI restriction enzyme cleavage sites were added at the 5 'and 3' ends, respectively. The prediction of secondary structures and stability of mRNA expressed in pET-32a(+) and pET-MOD were evaluated by the centerfold server. Additionally, the G+C content of these two fragments was evaluated by Rare Codon Analysis Tools. The designed gene was synthesized by the Biomatic Spa company. The designed pUC-57 gene carrier was cleaved by XbaI and XhoI enzymes to release the synthetic MOD fragment.
The pET-32a(+) vector was also cleaved by XbaI and XhoI enzymes and dephosphorylated by alkaline phosphatase to linearize the plasmid. The purification of linearized plasmid and MOD cut DNA was performed by agarose gel purification kit. The pET vector and the MOD extension fragment were joined by T4 DNA ligase to form the pET-MOD vector. The obtained vectors were heat-transfected to 100 μL of E.coli-BL21 (DE3) cells transfected using CaCl2; then, the bacteria were spread on a cell culture plate containing Luria-Bertani medium and 100 μg/mL ampicillin. They were incubated overnight at 37°C. After transformation, recombinant colonies were separated based on plasmid size. Furthermore, the positive colonies were examined for the presence of insert fragment using restriction enzyme cleavage analysis. The PCR colonization technique conducted the final confirmation of recombinant colonies, and the PCR product was confirmed by 1% agarose gel and DNA sequencing.


Results
Predicting mRNA secondary structure suggested that the optimization of the pET expression vector could prevent the formation of a stable secondary structure. The free energy of the second mRNA structure also changed from -122.8 to -20. Besides, the percentage of the G+C content of these two genes was similar and in the ideal range (50%-51%). After the enzymatic digestion of pUC-57, the 111bp fragment was released from the MOD synthesized gene; then, it was ligated into the XbaI and XhoI sequences and replaced with the 577bp sequence from the pET-32a(+) sequence.
In this optimized nucleotide sequence, genes expressing amino acid sequences, including Trx-Tag with His-Tag (thrombin protease enzyme digestion site), S-tag (TEV protease enzyme digestion site), and multiple cloning site (multiple cloning site) followed, were deleted. The pET-32a(+) vector length decreased from5900 bp to 5434bp (466bp decrease). Furthermore, the identification site of the restriction enzymes Sfi-I, BamH-I, and EcoR-I were replaced with the pET-MOD sequence. By the cleavage of the BamH-I and EcoR-I sequences, adherent sequences were created to bind the target protein gene with a similar tail. After the cleavage of the pET-MOD vector with Sfi-I, a linear plasmid with adherent ends consistent with the adherent end of the ELP gene cut by Bgl-I and pflM-I was generated. The nucleotide sequence encoding GGSGGSG (glycine + cysteine) was added to the MOD sequence, as a flexible linker region between the target protein sequence and the ELP tag.
Moreover, the WYWYW (tryptophan + tyrosine) coding nucleotide sequence was added to the MOD sequence to estimate recombinant protein concentration. The recombinant plasmid was purified from the transformed cell and identified based on plasmid size. Eventually, colony-PCR was performed to confirm the recombinant pET-MOD plasmid. Besides, the presence of 160bp band on 1% agarose gel and the sequencing of the resulting gene revealed the accuracy of cloning. The resulting pET-MOD plasmid sequence is available (code: KP834588.1).
Discussion
The production of large amounts of the bioactive fusion protein is a critical issue in biotechnology, and ELP fusion is an appropriate choice for this purpose [21]. The ELP tag, like many other tags, might reduce the bioactivity of the fusion protein based on the size and orientation of it [23]. Studies on the production of recombinant protein fusion and the ELP tag, have used the full-length synthesis of the target gene to put these genes together. As a result, that method will be costly, and the sequence development will be complex and time-consuming (due to its timely nature) [24].
 Alternatively, primary cloning in the non-expression vector, followed by sub-cloning, was applied. This method is also time-consuming, and because of the non-specificity of vectors, oligomerization will be complicated [25, 26]. The present study, for the first time, used the pET vectors and the MOD synthetic gene, to design and generate the pET-MOD specific vector in high volumes of the bioactive fusion protein with the oligomerization ability of the ELP tag. The synthetic MOD gene was designed based on the following requirements: 1. Reduce the sequence length of the pET-MOD vector by omitting the unnecessary sequence in the pET-32a(+) base vector; 2. Add Sfi-I locus sequence sequences that complement the inserted gene resulting from the oligomerization of the ELP gene generated by the RDL technique. Furthermore, it will reduce the complexity of the oligomerization of the ELP gene in non-specific vectors; 3. Incorporate the sequence identification of BamHI and EcoRI to the sequence integration and construction of fusion protein with ELP; 4. Incorporate a nucleotide sequence box encoding a flexible linker between the ELP tag and the target protein. This process helps to reduce the spatial interference on recombinant protein activity, particularly concerning its effect on the function of ELP5 tag fusion growth factors. The incorporation of the nucleotide sequence box encoding aromatic side-chain amino acids to absorb the UV light of the recombinant protein (to calculate protein concentration), particularly for proteins lacking aromatic amino acids [29].
Conclusion
The pET-MOD properties highlight the appropriate application of this vector for purifying recombinant protein and generating fusion protein with ELP tag (for use as a scaffold containing elastin as part of extracellular matrix). Furthermore, considering the presence of growth factors based on the study purpose, it is recommended for various studies, including wound healing research.
Ethical Considerations
Compliance with ethical guidelines
This study obtained its ethical approval form the Research Ethics Committee of Arak University of Medical Sciences (code: 92-146-11).

Funding
This study received financial support from the Deputy for Research and Technology of this university.
Authors' contributions
Investigation and initial draft preparation: Mohammad Reza Soleyman and Mostafa Khalili; Initial draft preparation and data analysis: Ali Reza Soleiman Meiguni; Review & editing, supervision, project administration: Maryam Baazm.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
The authors would like to thank the Deputy for Research and Technology of Arak University of Medical Sciences for their support.

References