Modares Journal of Biotechnology

Abstract Activin A, a member of the transforming growth factor-β (TGF-β) superfamily, plays a central role in numerous physiological processes such as cell differentiation, tissue repair, angiogenesis, differentiation of stem cells, cell adhesion and apoptosis. Because of its various clinical usages, recombinant production of it is beneficial. Since E. coli is one of the most popular hosts for recombinant protein production, in this study, cytoplasmic expression in this strain was used to produce high levels of Activin A. So, the cDNA of the Activin A mature region was amplified and then cloned in pET28a(+) vector. The resulting vector was transformed to BL21(DE3), BL21(DE3)plysS, and BL21(DE3)Rosetta-gami strains. After induction the promoter by using IPTG, Activin A production was confirmed by SDS-PAGE and Western blotting assays. The results showed that the expression of Activin A in the cytoplasm of all three strains was an efficient approach to obtain high levels of recombinant protein, but BL21(DE3) strain produced more protein. At the next step in order to achieve soluble form of Activin A, co-expression of cytoplasmic chaperones TF, GroEL/ES, and DnaK with pET28a (+) vector was used. The SDS-PAGE and Western blotting results showed that co-expression of Activin A with cytoplasmic plasmid pGro7 containing GroEL and GroES chaperones, in BL21(DE3) strain is an efficient approach for producing of soluble Activin A.

Abstract Escherichia coli is a Gram-negative bacterium, the second most common bacteria in the intestine and the main indicator of urban water pollution, is the most common cause of urinary tract infection and also is one of the main factors in food poisoning and diarrhea. Drug resistance of this bacterium to antibiotics is a global challenge. Horizontal gene transfer (HGT) is the movement of genetic material between unicellular and other gene transfer pathways which is an important factor in the evolution of many organisms, antibiotic resistance in bacteria, gene function. Antibiotic resistance in E. coli can be transferred to another species of bacteria through HGT mechanisms. Today, Bioinformatics methods have been used to understand of gene transfer from HGT mechanism. In this study, we used bioinformatics tools such as PredictBias, ACLAME, Mobil Genetic Elements (MGEs) PAI-ID, and Alien_Hunter in order to genes analysis that related from antibiotic resistance in E. coli. Bioinformatics and MIC assays result show that from 26 to 30 genes have been identified in all safthwers. Most of genes that identified show over 50 percent of GC content. put P gene with 178, blaCMY with 62, BlaTEM with 43, and aac-6 with 66 homology in the PredictBias website identified. Also in the ACLAME website, mob (A-C) and rep (A-C) gene family are highest number of horizontal gene transfer from infection bacteria strain. Those cluster genes are the highest resistant of laboratory tests which carries resistant genes such as blaSHV and blaCHV on the blaCMY plasmid.

Abstract Aims: There are several cell disruption methods for intracellular protein extraction. The aim of this study was to select the best approach for recombinant teriparatide fusion protein extraction from E. coli and achieve the best purification conditions.
Materials & Methods: In this experimental research, bacterial cells were disrupted by different methods such as sonication in different cycles, grinding with liquid nitrogen in two different cell culture volumes, and homogenization at two different pressures. The supernatant and pellet samples were run on sodium dodecyl sulphate gel. All the cell lysates were cultured on LB agar medium and stained with Gram staining method. The Ni²⁺ affinity chromatography of recombinant teriparatide fusion protein was done under denaturing and non-denaturing conditions, using pH and imidazole concentration gradient, respectively. All samples were taken on sodium dodecyl sulphate-polyacrylamide gel and the amount of purified protein was calculated by Micro-Bradford assay.
Findings: In the 20 and 25 cycles, a large part of the fusion protein led to protein solubilization. In the method of grinding with liquid nitrogen, proteins were more likely to enter the sediment part. The cell disruption was complete in a chemical method. The cell disruption under 50bar homogenization was more than that of 15bar. In chemical degradation and sonication, a large amount of fusion protein led to protein solubilization. In non-denaturing conditions, no recombinant fusion protein was removed from the column with the isolation buffer, but in the denaturing conditions, a large amount of proteins was purified.
Conclusion: The combined method of chemical degradation and sonication leads to approximately 97.7% of protein solubilization, and the purification in denaturing condition has also the suitable result in contrast to non-denaturing condition.

Abstract Aims: Nerve growth factor (β-NGF) is an important therapeutic agent for the treatment of neurodegenerative diseases such as Alzheimer’s disease; so, recombinant production of it in industrial scale is of high importance. The aim of this study is to optimize the effective factors in achieving the highest rate of β-NGF protein production in the bioreactor.
Materials & Methods: As E. coli is a suitable host for industrial production of recombinant proteins, E. coli DE₃ strain was used for production of recombinant β-NGF. Also, fermentation was performed in a 5-L bioreactor and % dissolved oxygen (%DO) and post-induction temperature values were optimized by response surface methodology (RSM). At first, the effects of these two variables on the level of total protein were studied. So, in every experiment, bacterial proteins were isolated and total protein concentration was determined by Bradford assay.
Findings: The results indicated that %DO and post-induction temperature of 30% and 28.5ºC were the best values for increased production of total protein; in these circumstances, total protein concentration was 9.6±0.61 mg/ml. Finally, the effects of these variables on recombinant β-NGF production were surveyed by dot blot analysis, indicating the maximum β-NGF expression level on the optimized condition.
Conclusion: In conclusion, %DO and post-induction temperature not only affect cell growth of recombinant E. coli, but also have a direct impact on recombinant protein expression and production, such as β-NGF.