Efficient process development of recombinant human granulocyte colony-stimulating factor [rh-GCSF] production in Escherichia coli

The protein hormone granulocyte colony-stimulating factor [GCSF] stimulates the production of white blood cells and plays an important role in medical treatment of cancer patients. An efficient process was developed for heterologous expression of the human GCSF in E. coli BL21 [DE3]. The feeding rate was adjusted to achieve the maximum attainable specific growth rate under critical value. In this method, specific growth rate was maintained at the maximum value of 0.55 h[-1] at the beginning of feeding to 0.4 h[-1] at the induction time. Recombinant human GCSF [rh-GCSF] was produced as inclusion body. At first, inclusion bodies were released by cell disruption and then washed, solubilized and refolded. Finally, the rh-GCSF was purified by cation exchange chromatography. Obviouly, higher specific growth rate decreases process time and consequently increases productivity. The final concentration of biomass and GCSF was achieved 126 g DCW.l[-1] and 32.1 g.l[-1]. Also, the final specific yield [Y[P/X]] and total productivity of rh-GCSF were obtained 254 mg.g[-1] DCW and 1.83 g.l[-1].h[-1], respectively. According to the available data, this is one of the highest Y[P/X] and productivity that has been reported for any human protein which is expressed in E. coli. Recovery yield of purification process was 40% and purity of recombinant protein was over than 99%. The circular dichroism spectra of purified rh-GCSF, Neupogen[registered] and PD-Grastim showed that all proteins have a similar secondary structure. Modified exponential feeding strategy for fed-batch cultivation of recombinant E. coli, results in minimum fed-batch duration and maximum productivity

Molecular cloning and characterization of the phenylalanine aminomutase gene from taxus baccata l.

Taxol is one of the most important anti-cancer drugs, which is obtained from yew trees [Taxus sp.]. T+he first step in side chain assembly of taxol is catalyzed by phenylalanine aminomutase, which converts alpha-phenylalanine to beta-phenylalanine. In this study, for the first time, we report on the cloning, preliminary expression and characterization of a full-length gene and cDNA encoding phenylalanine aminomutase from Taxus baccata L. Comparison of the full-length gene with other ones identified from the Taxus species showed high similarity, particularly with Taxus x media. The results showed that the expression level of this gene in Taxus baccata is very low and therefore this enzymatic step could be a rate limiting step in the taxol biosynthesis pathway. Successful amplification of the cDNA was only obtained from RNA samples isolated from methyl jasmonate elicited suspension cells of Taxus baccata. The cloned cDNA contained a 2064 bp open reading frame encoding a protein composed of 687 amino acids. Sequence comparison analysis revealed that the gene is very similar [98 - 99%] with respect to the nucleotide and amino acid sequences in different Taxus species and also share the signature active site motif [175ASG177]. The predicted structure of TbPAM was analyzed using bioinformatic tools. The results indicated that the protein has similar overall folding to tyrosine aminomutase.

Ligation independent cloning of polycistronic, genetically modified, HuMAb4D5-8 F [ab'] 2, in bacterial plasmid

In recent years, recombinant monoclonal antibodies and their derivatives have emerged as important targeted therapy agents. Monoclonal antibodies are ex-tremely difficult to produce. So, the cost of production is very high and many people cannot afford these drugs. In this regard, choosing inexpensive and easy ways to manipulate production systems such as bacterial hosts to reduce the cost of manufacturing these critical components are considered as vital step for developmental issues in recombinant expression systems. We, therefore, at-tempted to generate a polycistronic construct of anti HER-2 F[ab']2 fragment antibody for insertion in an expression bacterial plasmid. Also some modifica-tions were made in the hinge region to express antibody F[ab']2 fragment in its authentic form preventing from multiple varieties of disulfide bond formation. Finally, synthesized construct was cloned in pET-32 Ek/LIC vector without using restriction enzyme digestion or ligation reactions. The results of this study showed that modified F[ab']2 fragment was simply and successfully inserted in Escherichia coli [E.coli] using the Ligation Independent Cloning technology

Comparison of epothilone and taxol binding in yeast tubulin using molecular modeling

Microtubules are unique cytoskeletal structures that have structural subunits of alphabeta tubulin. Taxol is a typical microtubule stabilizing drug. The epothilones are other natural products with similar mechanism of action totaxol. Despite the highly conserved nature of beta-tubulin, some organism like Saccharomyces cerevesia is resistance to taxol, but sensitive to epothilones. In order to find differences in sensitivity of yeast tubulin to these molecules, we investigated binding mode of the taxol and epothilone A to yeast tubulin using molecular modeling. The multiple sequence alignment of beta-tubulin of different species was performed using ClustalW2. Protein structure of yeast beta-tubulin was constructed with Swiss Model 8.05 by using 1TVK. Modeled tubulin was superimposed with PyMol on 1JFF for comparison of three-dimensional structure of two proteins. Our results showed that one of the most interesting differences in binding mode of these molecules is residue 227. The His227 in bovine makes a hydrogen bond by means of its delta-nitrogen with epothilone A and by means of its epsilon-nitrogen with taxol. The Asn227 of yeast can play role of the delta-nitrogen of imidazole ring of H227, but not of epsilon-nitrogen of it. So yeast tubulin in contrast to taxol can interact with epothilone A. Due to conservation of essential residues for binding [T274, R282 and Q292], epothilone A in comparison with taxol can tolerate the interchange in the binding pocket [R276I]. Our findings may be of a great aid in the rational design of anti-tumor agents that bind to the taxol binding region of tubulin

[Creation of tenecteplase-producing CHO cell line using site-specific integrase from the phage phi C31]

The aim of this study was to produce a stable CHO cell line expressing tenecteplase. In the first step, the tenecteplase coding sequence was cloned in a pDB2 vector containing attB recognition sites for the phage phi C31 integrase. Then, using lipofection, the CHO cells were co-transfected with constructed recombinant plasmid encoding tenecteplase and attB recognition sites and the integrase coding sequence containing pCMV-Int plasmid. As the recombinant plasmid contained the neomycin resistance gene [neo], stable cells were then selected using G418 as an antibiotic. Stable transformed cells were assessed using genomic PCR and RT-PCR. Finally, the functionality of tenecteplase was evaluated on the cell culture media. our results indicated that tenecteplase coding sequence was inserted into the CHO cell genome and was successfully expressed. Moreover, tenecteplase activity assessment indicated the presence of our functional tenecteplase in the cell culture medium. Considering the data obtained from this study, phi C31 integrase can be used for the production of a stable cell line and it be used to introduce ectopic genes into mammalian cells