HPV16-E7 Protein T Cell Epitope Prediction and Global Therapeutic Peptide Vaccine Design Based on Human Leukocyte Antigen Frequency: An In-Silico Study.

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.

Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase.

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.

Overexpression of Recombinant Human Teriparatide, rhPTH (1-34) in Escherichia coli : An Innovative Gene Fusion Approach.

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) ß-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 (DH5α) 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 ß-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.

Down Regulation of ackA-pta Pathway in Escherichia coli BL21 (DE3): A Step Toward Optimized Recombinant Protein Expression System.

BACKGROUND: One of the most important problems in production of recombinant protein is to attain over-expression of the target gene and high cell density. In such conditions, the secondary metabolites of bacteria become toxic for the medium and cause cells to die. One of these aforementioned metabolites is acetate, which enormously accumulated in the medium, so that both cell and protein yields are affected. OBJECTIVES: To overcome this problem, several strategies applied. In this research we used antisense RNA strategy, where the transcription of phosphotransacetylase (PTA) and acetate kinase (ACK), two acetate pathway key enzymes, could be controlled, which led to reduced acetate production. MATERIALS AND METHODS: In order to achieve this, recombinant plasmid harboring antisense sequences targeting both of pta and ackA was assembled, after transfecting to the cells, its effects on the cell growth and acetate accumulation in the minimal media was assessed and compared with the control, the plasmid without antisense cassette, in presence and absence of IPTG in Escherichia coli BL21 (DE3). RESULTS: It was observed that the mentioned strategy partially affect the growth and amount of excreted acetate in comparison with the control. In addition it was found that high down-regulation of the acetate production pathway reduces the growth rate of E. coli BL21 (DE3). CONCLUSIONS: The study principally proved the importance of this strategy in acetate excretion control.