جستجوی مقالات مرتبط با کلیدواژه « expression optimization » در نشریات گروه « زیست شناسی »

Proteases are the most important industrial enzymes. Bacillus bacteria are commonly used to produce these enzymes. The aim of this study was cloning, sequencing, expression and bioinformatics study of aprX serine protease gene extracted from Bacillus licheniformis.

In this study, after extraction of bacterial DNA, aprX serine protease genes was isolated from Bacillus licheniformis and cloned into pTG19-T vector and then subcloned in pET28a vector. The molecular structure, its biochemical and phylogenetic properties were investigated and three-dimensional structure of the cloned enzyme was predicted. For the induction of gene expression and protein production of the recombinant serine protease IPTG was used at different concentrations, different temperatures and different time periods. Confirmation of aprX gene expression was performed by SDS-PAGE and dot blot analysis. Then, the activity of recombinant protease enzyme was measured at different temperatures and pHs.

Cloning was confirmed by sequencing. Based on the results of phylogenetic studies, the obtained protein sequence showed a high similarity to the sequences of other Bacillus species. After evaluating the drawn models, it was found that the models provided by RAPTROX and I-TASSER software were desirable models for predicting the three dimentional structure of this protease. The recombinant protein production was successfully induced by IPTG induction in the host containing the plasmid pET28a-aprX. The highest expression values were obtained at 25 ° C for 20 hours with 0/5 mM IPTG. Also, the recombinant protein produced showed the highest activity at 50 ° C and pH 8.

Cervical cancer is the leading cause of morbidity and cancer mortality in women throughout the world and persistent infection with human papillomavirus (HPV) is the main etiology for the development of this cancer. Peptide vaccines derived from E6 and E7 oncogenes are promising candidates for HPV vaccine production due to their safety, high stability, and ease of production. In the current study, expression strains such as Escherichia coli BL21 (DE3), BL21 (DE3)-AI, Rosetta, C41 (DE3), and BL21 (DE3)-pLysS were applied for poly epitopic E6 expression and the effects of temperature, induction time, and inducer concentration on the expression level of recombinant HPV16-E6 polypeptide protein were examined in the mentioned strains.

In the present study, the optimized gene of E6 recombinant protein was cloned into pET28a vector under the control of T7 promoter and the resulting plasmid was successfully transformed into Escherichia coli strains BL21 (DE3), BL21 (DE3)-AI, Rosetta, C41 (DE3), and BL21 (DE3)-pLysS. Then, the ability of these strains to express the desired recombinant protein in different conditions was compared.  Two temperatures of 25 ° C and 37 ° C, IPTG concentrations between 0.1 and 1.0 mM, and different incubation times were selected to examine the expression level of recombinant E6 protein in these strains.

The highest level of expression was obtained in Escherichia coli BL21 (DE3) -AI strain inducing at Optical Density (OD) of 0.9, 0.1 mM IPTG, and 16 hours induction at 25 °C.

The results of this study can be used to produce E6 protein as a vaccine candidate in the treatment of HPV-related cancers.

Uropathogenic Escherichia coli (UPEC) is one of the most common bacteria to cause urinary tract infection (UTI). No human vaccine against UTI has yet been developed. The aim of this study was to optimize the expression of recombinant PapG with Lactobacillus anchor protein AcmA in E. coli.
The synthetic cloning vector, pEXA containing PapG.AcmA was purchased and subcloned into pET21a vector. The protein expression levels in Origami expression host (E. coli) were analyzed by SDS-PAGE gel and western blotting. Moreover, various concentrations of IPTG (Isopropyl Thiogalactopyranoside), the medium component and induction time was optimized for large scale expression of recombinant protein.
Based on results, optimum expression in large scale was occurred in 0.1mM IPTG and OD= 3 optical density. The modified complex culture medium containing: glucose 6 g/I, K2HPO4 12.5 g/l, KH2PO4 2.3 g/l, Yeast Extract 20 g/l, tryptone 10 g/l were determined as optimal medium. OD 600nm= 3.0 was determined as the best time for induction by IPTG at a concentration of 0.1 mM. The levels of the expression of the target protein was determined at OD600nm= 5.5.
Based on the result, we were able to do cloning and expression of PapG.AcmA. Addition of extra carbon source (glucose) to the complex medium caused a better PapG.AcmA recombinant protein expression. Finally, by purification of recombinant protein and evaluation of its immunogenicity, it can be used as a vaccine candidate against the urinary tract infection.