Many of biopharmaceutical protein products and industrial enzymes such as monoclonal antibodies and antibody fragments, blood clotting factors, thrombolytic agents, vaccines, hormones, interferons, interleukin-based products, growth factors, nucleic-acid based products, and therapeutic enzymes are recombinant proteins (14-16). Appropriate and efficient strategies for the production of high amounts and high-quality of recombinant proteins are important. Several factors affect the expression of recombinant protein, one of the most important factors that can increase the concentration of target protein is selection of the appropriate expression host. From conventional expression systems, prokaryotic, yeast, insects, and mammals, the E. coli expression system is one of a well-established system. The main causes for selection of the E. coli expression system is due to the ability to grow rapidly, low cost culture conditions, easy genetic manipulation, high expression levels of target gene, accessibility of versatile plasmid vector, a well-knowledge of biochemical, and genetic characterizations (2, 5, 6). A variety of cloning and expression vectors are available for production of recombinant proteins. One of the most powerful and very popular systems that is widely used for cloning and expression of recombinant proteins in E. coli is the pET system. The pET system utilizes a strong promoter (bacteriophage T7 promoter), in which the target gene is cloned behind the T7 promoter and expression induced by the T7 polymerase RNA in the host source. In successful cases and a few hours after induction, the desired protein can represent 50% of the total cell protein. Another important benefit of this system is that in the non-induce state, the transcription of the desired gene becomes silent (1, 3, 4). The E. coli expression system has common features with other vectors, including an origin of replication, promoter, a selection marker (e.g. antibiotic resistance gene), translation initiation site, 50 untranslated region (50UTR), and N-terminal codons. Unlike other elements mentioned above, fusion tags in the expression vectors can be expressed along with the target gene, which it can along with other factors; promoters, 50UTR, N-terminal codons have a significant effect on transcription, protein yields, solubility, and purification. Different tags are used to facilitate the expression and purification of recombinant proteins in host Escherichia coli, including Thioredoxin (Trx), 6xHIS-Tag, small ubiquitin-like modifier (SUMO), glutathione S-transferase (GST), green fluorescent protein (GFP), HaloTag, and maltose binding protein (MBP). Among these, due to some of the properties such as commercially available expression vectors and their downstream processing systems and more than 80% purity can be captured in a one chromatographic phase, the 6xHIS-Tag as one of the most popular tags is used (3-5). Different approaches can be used for cloning of the heterologous gene in suitable vector such as restriction enzyme (RE)-based cloning, recombination-based cloning, and annealing-based or ligation-independent cloning (LIC). Researchers prefer to use the restriction enzyme (RE)-based cloning technique compared to traditional digestion–ligation methods, due to several advantages including simple, efficient, easier to set up, universal and cost-effective. Therefore, in this study, RE-based cloning method was applied (6). After amplification of the heterologous gene using specific primers, ligation process (ligation of the heterologous gene and vector) was done by ligase enzyme and then, transformed into E. coli competent cells. Therefore, we were able to express and produce high levels of fliC-NT300 fusion protein in the pET28a system under the powerful T7 promoter. In addition, due to well-known genetics, cheap, and fast cultivation of E. coli BL21 (DE3), this system is used as a potent and versatile system (2, 3, 5). SDS-PAGE results showed that recombinant fusion protein was considerably expressed in cell lysate to compare with non-induced samples. In addition, the presence of the C-terminal 6xHIS-Tag, in the fusion protein, could help detect fusion protein in Western Blot analysis using anti-his conjugate-antibody followed by purification of fusion protein using Ni-NTA affinity column.
4.1. Conclusions
Amplification, cloning, and expression of fliC-NT300 fusion protein were carried in E. coli host, successfully. In addition, it is essential that the other studies, including in vitro and in vivo, be carried out on this recombinant protein in the future.
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