zahra hajihassan; Navid Nazari
Abstract
Nowadays recombinant activin A has been produced in different expression hosts because of its vast clinical applications. As obtaining the highest cell density is one way to increase the production, optimization of cell culture medium is necessary. So in this study, glycerol and yeast extract concentration ...
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Nowadays recombinant activin A has been produced in different expression hosts because of its vast clinical applications. As obtaining the highest cell density is one way to increase the production, optimization of cell culture medium is necessary. So in this study, glycerol and yeast extract concentration as carbon and nitrogen sources of medium was optimized by using RSM (response surface methodology) in order to achieve the highest cell density of E. coli DE3) 21BL) strain transformed with pET21a:activin A vector. Furthermore, the effect of MgCl2 on the growth of mentioned bacterium was studied. The results showed that the highest cell density (growth) was achieved in 42.5 g/L of yeast extract, 22.5 g/L of glycerol and 30 mM of MgCl2. Also, dot blot results and data analysis with Image J in this study showed that the expression level of recombinant activin A in the optimized medium has been increased in comparison to standard cell culture medium.
Masoumeh Rajabi bazl; zahra Hajihassan; Mona Sohrabi
Abstract
Beta Nerve growth factor (β-NGF) was first known for its vital role in development and survival of neurons. This protein, which belongs to the neurotrophin family, plays a considerable role in the treatment of neurodegenerative diseases such as Alzheimer’s disease (AD). As a result of its ...
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Beta Nerve growth factor (β-NGF) was first known for its vital role in development and survival of neurons. This protein, which belongs to the neurotrophin family, plays a considerable role in the treatment of neurodegenerative diseases such as Alzheimer’s disease (AD). As a result of its characteristic, recombinant human β-NGF (rhβNGF) was expressed in two different strains of E.coli for the first time in Iran. β-NGF has three disulfide bonds in its native and functional structure, so the cytoplasmic reducing environment of E.coli is not appropriate for its expression. Therefore, the oxidative space of periplasm for production of correctly folded β-NGF was considered. In this study, hβNGF cDNA obtained from NCBI data bank after codon optimization and subcloning in pET39b(+) vector containing DsbA gene was transformed (using heat shock) to BL21(DE3)pLysS and BL21(DE3) strains of E.coli. Co-expression occurred via induction of promoter with 1 mM of IPTG. Consequently, extracted proteins from these two strains were compared with each other with SDS-PAGE and Dot blot techniques. The data shows βNGF expression especially in BL21(DE3) strain of E.coli.
zahra Hajihassan; Azra Rabbani-Chadegani
Abstract
Actinomycin D and mitoxantrone are anticancer drugs widely used in the treatment of a variety of cancers. DNA has been introduced as a main target for these drugs which results in inhibition of both DNA replication and RNA transcription. In the cell nucleus, DNA is not naked but is complexed with histone ...
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Actinomycin D and mitoxantrone are anticancer drugs widely used in the treatment of a variety of cancers. DNA has been introduced as a main target for these drugs which results in inhibition of both DNA replication and RNA transcription. In the cell nucleus, DNA is not naked but is complexed with histone proteins producing a compact structure called chromatin. In the present study we have investigated and compared the binding affinity of these drugs to rat liver chromatin. The results of gel electrophoresis and UV-Vis spectroscopy showed that both drugs bound to chromatin but mitoxantrone exhibited higher affinity compared to actinomycin D. Disappearance of histones on the gel and reduction of the absorbencies at 608 and 440 nm, corresponding mitoxantrone and actinomycin D respectively, suggest compaction/aggregation of the chromatin. The binding isotherms demonstrated a positive cooperative binding pattern for both drugs with binding constants (ka) of 7.1×106 M-1 and 0.35×105 M-1 for mitoxantrone and actinomycin D respectively suggesting higher binding affinity of mitoxantrone to chromatin compared to actinomycin D. In addition Gibbs free energy for the binding of both drugs was negative demonstrating that the reaction is spontaneously. It is concluded that actinomycin D preferentially interacts with DNA molecule, whereas, mitoxantrone binds to nucleoprotein structure of chromatin implying the role of histone proteins in this binding process.
