Mahsa Rezaee, Fahimeh Baghbani Arani, Reza Arabi Mianroodi,
Volume 3, Issue 3 (12-2016)
Abstract
Streptokinase is one of the best known thrombolytic agents with widespread clinical use. However, its use is not risk-free due to its immunogenicity, hemorrhagic complications and relatively short half-life in circulation. Specific PEGylation of cysteine residue is a useful technique for reducing most of these complications. The aim of this study was designing and producing a cysteine containing mutant of streptokinase, to be used for specific PEGylation. Glut-amic acid 263, which is a surface amino acid in the structure of streptokinase protein, was selected for replacement with cysteine amino acid by site directed mutagenesis. The Glu263 codon was changed to cysteine codon by SOEing PCR technique. Then, the intact and mutated streptokinase genes were inserted into expression vector pET-26b (+). The co-nstructs were transformed to Escherichia.coli Rosetta (DE3) strain and the proteins were expressed by IPTG induction. The proteins were confirmed by SDS-PAGE and western blot analysis, purified by Ni-NTA agarose affinity chroma-tography under denaturing condition with urea and Sephadex G-25 column was applied to remove urea to refold the pr-oteins. This study indicated that by using aforesaid vector and host, cysteine containing mutant gene is expressed well and it will be appropriate for specific PEGylation.
Lida Jalali Roudsary, Azarnoosh Jafari, Jamil Vaezi, Ehsan Karimi,
Volume 9, Issue 2 (9-2022)
Abstract
High-quality DNA extraction plays an important role to make sharp bands in the gel electrophoresis and also produces clean chromatograms. Usually, DNA extract is delivered using the modified CTAB method but this method cannot obtain high-quality DNA for molecular analysis from old dried leaves of Bellevalia due to having different chemical compounds which inhibit to obtain a clear DNA extraction. To solve the problem, in different phases of a modified CTAB protocol, activated carbon, phenol, potassium acetate, RNase and deionized water (instead of TE) were applied to eliminate contaminants and improve obtained high-quality DNA. The DNA absorption data from the modified method were compared with CTAB method in 260 and 280 nm wavelengths. The results showed a 15-80-fold change in the DNA concentration. This study suggests the modified method of extraction is more efficient in the quality of DNA obtained from the little amount of herbarium old leaves of Bellevalia than that of the CTAB method.
Mahdi Alijanianzadeh, Alireza Jalalvand, Rasoul Khalilzadeh, Maryam Abdolirad,
Volume 9, Issue 4 (3-2023)
Abstract
S-layer proteins of Deinococcus radiodurans are the best self-assemble systems among other proteins that have an essential role in the fabrication of nanowires. Therefore, the purification of these proteins is necessary. The purpose of this research was to optimize the purification of s-layer protein from D. radiodurans with the response surface method. The three factors of SDS concentration, incubation time and mass percent in five levels were considered, and 20 runs were designed by Design-Expert software with a central composite method. Each run includes microbe culture, mass cell preparation, microbe incubation in specific SDS concentration and time and mass percent, separation of the bacteria from detergent with a centrifuge at 5000g, sedimentation of s-layer proteins from detergent solution with a centrifuge at 20000g, determination of protein concentration, and protein purity by Bradford and SDS-PAGE methods, respectively. Finally, the data obtained were analyzed. Analysis of the results demonstrated that at the 95% confidence level, the effect of the detergent concentration factor on the purified protein percent was more than other factors. The optimization results of factors are 5.64% SDS concentration, 7.33% mass percent, and 3 hours incubation time. At optimized conditions the protein concentration and purity percent were obtained 0.584 mg/ml and 47.61% respectively.
