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Human activin A is a homodimer of βA subunit which is synthesized in the form of prepro-activin with 426 amino acids; mature activin A with 116 amino acids is processed from this larger precursor protein. This protein which was extracted for the first time from follicular fluid is a strong stimulator of FSH biosynthesis. The functions have been found to be exerted by activin, including roles in cell proliferation, differentiation, apoptosis and survival of neurons. As this protein plays a considerable role in the treatment of neurodegenerative disease such as Alzheimer,s disease and wound repair, in this study for the first time was expressed in three different strains of E.coli. Activin A has 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 activin A was considered. In this study, h-activin A cDNA and modified Iranian Bacillus Licheniformis α-amylase signal peptide obtained from NCBI data bank after codon optimization was cloned in pET21b(+) vector and transformed to BL21(DE3)pLysS, BL21(DE3)Rosetta gami and BL21(DE3) strains of E.coli. Expression occurred via induction of promoter with IPTG. Consequently, extracted proteins from these three strains were compared with each other using SDS-PAGE, Dot blot and western blot techniques. The data shows activin A expression especially in BL21(DE3) and BL21(DE3)Rosetta gami strains of E.coli.

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Purpose: In order to express human granulocyte-macrophage colony stimulating factor (hGM-CSF) under heat shock. Materials and Methods: Two expression plasmids were constructed based on pBC(SK) plasmid. The expression cassettes in the two plasmids are equipped with a 75 base pair fragment, derived from the PL promoter of the bacteriophage lambda (λ). The plasmids also contain a temperature mutant of repressor coding gene (CI857) to regulate the promoter activity. The two plasmids differ from each other in having a transcription termination signal or not, down stream to the recombinant gene in the expression cassette. A pelB signal sequence was also used in order to have the recombinant protein in the periplasmic space of Escherichia coli. The efficiency of the constructed plasmids was demonstrated by heat-regulated expression of hGM-CSF. Results and Discussion: The protein analysis of the recombinant bacteria, containing either of the two plasmids, indicates a successful expression and complete processing of the hGM-CSF precursor, following the heat shock activation of the λPL promoter. In order to enhance the applicability of the terminator containing plasmid, for the expression of other proteins of interest by heat regulation, a multiple cloning site including eleven unique restriction sites was inserted in the plasmids. The heatregulated plasmids, designed in this work, have provided suitable tools to study the expression of recombinant proteins under temperature up-shift in Escherichia coli, when the use of chemical inducers are not desirable.

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Hyper-glycosylation is an approach to introduce new N-glycosylation consensus sequence(s) (َAsn-Xxx-Ser/Thr three-peptide) into a protein primary amino acid sequences by site-directed mutagenesis which is followed by the attachment of a new glycan to the Asn residue located within the three-peptide sequence. Hyper-glycosylation has attracted lots of interest especially in the protein therapeutics industry. The attached glycan may improve the pharmacokinetic properties of the hyper-glycosylated priteins and increase their half-life in the bloodstream. In the current study, a new N-glycosylation site was introduced into N-terminal Gla domain of hFIX. Arg³⁷ position of mature hFIX was targeted to be converted into Asn residue by site-directed mutagenesis using overlap extension PCR. Recombinant expression plasmids for native and mutant hFIX were constructed. The expression of the recombinant wild-type and mutant hFIX was analyzed in mammalian HEK293 cells using gradient SDS-PAGE and western blotting analysis. The results indicated in higher molecular weight for R37N mutant in compared with the native protein. The glycan attachment to R37N mutant was further confirmed by PNGase digestion and western blotting. 

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Objective: Eukaryotic proteins generally have signal peptides which are not only crucial for their secretion efficiencies but are important for their expression levels. Coagulation factor IX (FIX) is a glycoprotein that plays a fundamental role in the blood coagulation pathway. Reduced levels or dysfunctional FIX are associated with hemophilia B. This study investigates the function of the human prothrombin signal peptide in an attempt to improve the human FIX (hFIX) secretion efficiency in a heterologous expression system. With this aim, we have used the SignalP and PrediSi programs for in silico evaluation of the signal peptide efficiency prior to conducting this experiment. Methods: We used molecular techniques to amplify and join the coding region of the human prothrombin signal peptide to the cDNA of mature hFIX. The chimeric fragment was examined for transient expression in a mammalian cell line (HEK293T) in comparison with the native hFIX, under a CMVp regulation. Using the neural network-based prediction programs, we evaluated the scores for cleavage position and secretion efficiency of the human prothrombin and hFIX signal peptides. The expression efficiencies of hFIX expressed by the recombinant cells were analyzed by RT-PCR and ELISA. Results: In silico analysis more efficiently predicted the human prothrombin signal peptide with a high score compared to the native hFIX signal peptide. This data was confirmed by the RT-PCR and ELISA results obtained from expression analyses at the RNA and protein levels, respectively. Conclusion: The present study showed that the signal peptide derived from the human prothrombin has the potential for efficient secretion of hFIX as evidenced by the results taken from a transient expression system. The results were consistent with in silico analysis. This replacement could be evaluated in a stable state condition.