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Cervical cancer is the second most common leading cause of women's death due to cancer worldwide, about 528,000 patients' cases and 266,000 deaths per year, related to human papillomavirus (HPV). Peptide-based vaccines being safe, stable, and easy to produce have demonstrated great potential to develop therapeutic HPV vaccine. In this study, the major histocompatibility complex (MHC) class I, class II T cell epitopes of HPV16-E7 were predicted. Therefore, we designed a plan to find the most effective peptides to prompt appropriate immune responses. For this purpose, retrieving protein sequences, conserved region identification, phylogenic tree construction, T cell epitope prediction, epitope-predicted population coverage calculation, and molecular docking were performed consecutively and most effective immune response prompting peptides were selected. Based on different tools index, six CD8+ T cells and six CD4+ epitopes were chosen. This combination of 12 epitopes created a putative global vaccine with a 95.06% population coverage. These identified peptides can be employed further for peptide analysis and can be used as a peptide or poly-epitope candidates for therapeutic vaccine studies to treat HPV-associated cancers.

Human follicle-stimulating hormone (hFSH) is a heterodimeric glycoprotein consisting of a common α subunit non-covalently attached to the hormone-specific β subunit. The therapeutic application of this hormone requires large quantities of biologically active contaminant-free hormones. In the current study, secretory expression and purification of codon-optimized recombinant α and β subunits of hFSH were reported using Iranian Lizard Leishmania (I.L.L.) expression system. Synthetic codon-optimized genes of α and β subunits of hFSH were amplified by PCR and cloned into the pLEXSY-hyg2 and pLEXSY-neo2 vectors, respectively. The resulted expression vectors were digested and electroporated into ILL. PCR analysis showed that the genes of α and β subunits of hFSH were inserted into the genome of the parasite. Subunits expression was analyzed by dot-blot and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and then purified and detected by Western blot analysis. Expression analysis confirmed the presence of protein bands in sizes similar to those of the natural hormone subunits. The low electrophoretic mobility and the apparent higher molecular weight of the expressed subunits were due to successful glycosylation of subunits by ILL expression system.

Several human diseases arise from abnormality in functional proteins production in different tissues. Recombinant protein drugs produced in special hosts are the most important solution for this problem. Efficient extraction and purification of recombinant protein from the host proteins is the fundamental stage in recombinant drug manufacture process. Teriparatide, a 34 amino acid peptide, is recombinant FDA approved drug used for osteoporosis stimulates the bone formation and rises its mass. In this research, 6his- E. coli K12β-gal (236-288)-hPTH (1-34) fusion protein was expressed by E. coli BL21 (DE3), extracted with combined chemical and sonication approach led to within 97.7 7% total proteins solubilization. Purification was done during two step chromatography and one enzymatic cleavage for rhPTH (1-34) separation. Approximately, 350 mg of fusion protein was obtained from 1 l culture volume after Ni2+ affinity. Enterokinase enzyme was used for fusion protein cleavage and recombinant teriparatide (1-34) was purified by cation exchange HiTrap SP XL chromatography column. Concentration of purified rhPTH (1-34) was estimated about 105 mg/1 l culture. Biological activity of rhPTH (1-34) was analyzed with serum calcium measurement in Vistar rat. The results were shown the rhPTH (1-34) was purified in this research has biological activity as equal as commercial drug. Efficient purification of active rhPTH (1-34) during the two step simple chromatography method is very precious in the industry of recombinant protein production.

Background: The potential of plants, as a safe and eukaryotic system, is considered in the production of recombinant therapeutic human protein today; but the expression level of heterologous proteins is limited by the post-transcriptional gene silencing (PTGS) response in this new technology. The use of viral suppressors of gene silencing can prevent PTGS and improve transient expression level of foreign proteins. In this study, we investigated the effect of p19 silencing suppressor on recombinant human nerve growth factor expression in Nicotiana benthamiana. Materials and Methods: The p19 coding region was inserted in the pCAMBIA using NcoI and BstEII recognition sites. Also, the cloned synthesized recombinant human NGF (rhNGF) fragment was cloned directly into PVX vector by ClaI and SalI restriction enzymes. The co-agroinfiltration of rhNGF with p19 viral suppressor of gene silencing was evaluated by dot-blot and SDS-PAGE. The amount of expressed rhNGF protein was calculated by AlphaEaseFC software. Results: Co-agroinfiltration of hNGF with P19 suppressor showed about forty-fold increase (8% total soluble protein (TSP)) when compared to the absence of P19 suppressor (0.2%TSP). Conclusion: The results presented here confirmed that the use of P19 gene silencing suppressor derived from tomato bushy stunt virus (TBSV) could efficiently increase the transient expression of recombinant proteins in Nicotiana benthamiana manifold.

Background: Pivotal roles of Nerve growth factor (NGF) in the development and survival of both neuronal and non-neuronal cells indicate its potential for the treatment of neurodegenerative diseases. However, investigation of NGF deficits in different diseases requires the availability of properly folded human β-NGF. In previous studies bacterial expression of hNGF demonstrated the feasibility of its overproduction. However, known limitations in the use of E. coli as an expression host for a protein with three intra-chain disulfide bonds were evident. Objectives: Here an optimized system was developed to overexpress the soluble NGF in E. coli.Materials and Methods: The gene encoding the β subunit of mature hNGF was optimized based on E. coli codon preference and cloned into pET-32a expression vector providing His- and Trx- tags for detection and increasing the solubility of recombinant protein, respectively. The recombinant DNA was expressed in E. coli Origami (DE3), which enhances the correct formation of disulfide bonds in the cytoplasm of E. coli. Different culture conditions were evaluated to increase soluble expression of the target protein.Results: The highest soluble expression level was achieved when E. coli Origami (DE3) cells expressing NGF were grown at 30oC in TB medium with 0.2 mM IPTG induction at OD600nm = 1 for 4 h.Conclusions: Our results indicated that the recombinant NGF was successfully expressed as a soluble form.

Background: Pseudomonas putida (P. putida) ATCC12633 can produce creatinase. It is a microbial enzyme which degrades creatinine in bacteria and provides source of carbon and nitrogen. Also, this enzyme is used in the enzymatic measurement of creatinine concentration for diagnosis of renal and muscles functions and diseases. Our purpose was recombinant production of creatinase for using in clinical measurement of serum or urine creatinine. Methods: A 1209bp of open reading frame of creatinase was amplified by PCR from P. putida ATCC12633 genome and cloned into pET28a expression vector which was digested using NheI and XhoI restriction enzymes. Cloning was confirmed by colony PCR, double digestion analysis and sequencing. Recombinant pET28a vector was transformed to Escherichia coli (E. coli) BL21 (DE3). Creatinase expression was induced in E.coli BL21 (DE3) using IPTG and confirmed by SDS-PAGE and western blotting. Purification of creatinase was performed using Ni-NTA column. The specific activity of this enzyme was also investigated. Results: The creatinase gene cloning was confirmed by DNA sequencing. Successful expression of creatinase was performed in E. coli (57.4% of total protein). SDS-PAGE and western blot analysis showed a 45 kDa creatinase protein. Purification of creatinase was done with high purity. The specific activity of recombinant enzyme is 26.54 unit/mg that is much higher than other creatinase used in the commercial kits (9 unit/mg). Conclusion: The P. putida ATCC12633 recombinant creatinase was expressed efficiently in E. coli BL21 and 57% of total protein was the recombinant creatinase. Also, expressed creatinase has high solubility and also the enzyme has good activity compared to enzymes used in commercial kits, so a new source of creatinase was produced for creatinine assay kit in this study.

Pivotal roles of Nerve growth factor (NGF) in the development and survival of both neuronal and non-neuronal cells indicate its potential for the treatment of neurodegenerative diseases. However, investigation of NGF deficits in different diseases requires the availability of properly folded human β-NGF. In previous studies bacterial expression of hNGF demonstrated the feasibility of its overproduction. However, known limitations in the use ofHere an optimized system was developed to overexpress the soluble NGF inThe gene encoding the β subunit of mature hNGF was optimized based onThe highest soluble expression level was achieved whenOur results indicated that the recombinant NGF was successfully expressed as a soluble form.

Background: Parathyroid hormone is an 84-amino acid peptide secreted by the parathyroid glands. Its physiological role is maintenance of normal serum calcium level and bone remodeling. Biological activity of this hormone is related to N-terminal 1–34 amino acids. The recombinant form of hormone (1–34) has been approved for treatment of osteoporosis from 2002. In this study, a novel fusion partner has been developed for preparation of high yield recombinant 1–34 amino acids of hPTH. Methods: Novel nucleotide cassette designed encoding a chimeric fusion protein comprising of a fusion partner consisting of a His-tag in N-terminal, 53 amino acids belong to Escherichia coli (E. coli) β-galactosidase (LacZ) gene, a linker sequence for increasing of expression and protection of target peptide structure from fusion tag effect, an Enteropeptidase cleavage site, rhPTH (1–34) gene fragment. Optimized fusion gene was synthesized and ligated into pET-28a vector under control of T7 promoter, and then transformed in E. coli (DHα) cells. Positive clones containing this gene were double digested with NcoI and-BamHI and also approved by sequencing. Gene overexpression was observed in SDS-PAGE after induction with 0.2 mM IPTG. Confirmation of gene expression was performed by western blotting using anti-His-tag antibody conjugated with peroxidase. Results: By this fusion gene design approach, we achieved a high level expression of the rhPTH, where it represented at least 43.7% of the total protein as determined by SDS-PAGE and confirmed by western blotting. Conclusion: In addition to high level expression of the designed gene in this work, specific amino acid sequence of bacterial β-galactosidase was selected as major part of carrier tag for protection of this hormone as important step of recombinant rhPTH with relevant isoelectronic point (pI). This innovation resulted in recombinant production of hPTH very well and the gene construct could be applied as a pattern for similar recombinant peptides where recombinant protein degradation is a critical issue.

Bioluminescence reaction, which uses luciferin, Mg(2+)-ATP and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by the luciferase and emit visible light. One of the most promising applications of firefly luciferase is biosensors. In order to develop an apoptosis biosensor based on caspase 3/7, we have generated 3 forms of circularly permuted variants of Photinus pyralis firefly luciferase and a relatively good tolerance toward disruption of the polypeptide chain by introduction of new termini were found. Two forms of circular permuted luciferases showed significant activity enhancement in comparison with control after exposure to caspase 3. Moreover, the effect of circular permutation and also the length of inserted peptide (caspase 3/7 recognition sites) in structure of firefly luciferase were analyzed using circular dichroism and fluorescence spectroscopy.