Comparison of three Escherichia coli Strains in recombinant production of reteplase

Background: Escherichia coli [E. coli] is the most extensively used host for the production of recombinant proteins. However, most of the eukaryotic proteins are typicallyobtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. Reteplase as a highly disulfide-bonded recombinant protein is an example of difficult to express protein in E. coli

Methods: In this study, a codon optimized reteplase gene was synthetically prepared and cloned under the control of an IPTG inducible T7 promoter. The vector was simultaneously transformed and expressed in three different E. coli strains. The ability of strains for expression of this recombinant pharmaceutical was compared. Also, an attempt was made to increase the soluble production of reteplase in SHuffle T7 E. coli with alterations of expression condition like temperature, inducer concentration and oxygen supply

Results: High amounts of reteplase were expressed as inclusion bodies in all three strains. BL21 [DE3] showed the highest level of expression in inclusion bodies followed by Rosetta-gami [DE3] and Shuffle T7. Changes of expression conditions were insufficient for soluble expression of reteplase in SHuffle T7 as a genetically engineered host for production of disulfide bonded proteins

Conclusion: The oxidizing cytoplasm of Rosetta-gami and Shuffle T7 in addition to alterations of cultivation parameters could not result in soluble production of reteplase, although the inclusion bodies produced in these two strains might increase the rate of refolding procedure likely due to formation of folding intermediates

Overexpression of recombinant human beta interferon [rhINF-beta] in periplasmic space of Escherichia coli

Human Interferon beta [INF-beta] is a member of cytokines family which different studies have shown its immunomodulatory and antiviral activities. In this study an expression vector was designed and constructed for expression of human INF-beta-1b either in shake flasks or bench top bioreactor. The designed vector was constructed based upon pET-25b[+] with T7 promoter. Recombinant human beta interferon [rhINF-beta] was codon optimized and overexpressed as a soluble, N-terminal pelB fusion protein and secreted into the periplasmic space of Escherichia coli BL21 [DE3]. The sugar, Isopropyl-beta-D-thiogalactopyranoside [IPTG] was used as a chemical inducer for rhINF-beta production in the shake flasks and bench top bioreactor. Timing of beta interferon expression was controlled by using the T7 promoter. The rhINF-beta protein was extracted from periplasmic space by osmotic shock treatment and the expression of the beta interferon encoding gene in random selected transformants, was confirmed by western and dot blot methods. The maximum of product formation achieved at the OD[600nm] = 3.42 was found to be 35% of the total protein content of the strain which translates to 0.32 g L[-1]. The constructed vector could efficiently overexpress the rhINF-beta into the periplasmic space of E. coli. The obtained yield of the produced rhINF-beta was more than previous reports. The system is easily adapted to include other vectors, tags or fusions and therefore has the potential to be broadly applicable to express other recombinant proteins

Maximizing production of human interferon- gamma in HCDC of recombinant E. coli

Tuning recombinant protein expression is an approach which can be successfully employed for increasing the yield of recombinant protein production in high cell density cultures. On the other hand, most of the previous results reported the optimization induction conditions during batch and continuous culture of recombinant E. coli, and consequently fed-batch culture have received less attention. Hence, in this research induction conditions for the over-production of recombinant interferon- gamma including the amount of inducer, induction time and post-induction duration during chemical induction were optimized. E. coli BL21 [DE3] [pET3a-hifn gamma] was used to over-express human interferon-gamma [hIFN- gamma] in an exponential fed-batch procedure with a maximum attainable specific growth rate of 0.55 h[-1] at the beginning of feeding and 0.4 h[-1] in induction time. The factors were considered as the amount of inducer [IPTG] in the range of 0.565- 22 mg g[-1] L[-1] at seven levels, cell density at induction time as 53, 65 and 75 g [dry cell weight] L[-1], induction duration at different intervals of 3, 4, and 5 h after induction time. The final concentration of biomass and interferon gamma reached to 127 g L[-1] [DCW] and 51 g [hIFN-gamma] L[-1] after 17 h, and also the final specific yield and overall productivity were obtained 0.4 g [hIFN- gamma] g[-1] DCW and 3 g [hIFN- gamma] L[-1] h[-1], respectively, which are the highest amounts of reported specific yield and productivity for recombinant proteins production

[Inhibition of ackA and pta genes using two specific antisense RNAs reduced acetate accumulation in batch fermentation of E. coli BL21 [DE3]]

Expression of foreign proteins in E. coli is normally inhibited by exogenous production of acetate. To overcome this problem, various strategies have been proposed and tested to reduce the extent of acetate accumulation. Although these strategies can improve the outcome, the implementation of their proposed techniques is not practical. Because to achieve optimal results, it requires extremely tight control conditions and the actual cost is very high. Furthermore, a simple knockout mutation of the target metabolic pathway would not be appropriate because the acetate pathway plays an important physiological role in E. coli. In this study, we employed an antisense RNA strategy as an elaborated metabolic engineering tool to partially block biosynthesis of two major acetate pathway enzymes, acetate kinase [ACK] and phosphotransacetylase [PTA]. The fragments of antisense cassette were cloned sequentially in pBluescriptsk+ and completed cassette subcloned in pLT10T3. The function of this cassette was evaluated with RT-PCR and ACK and PTA assay. The effect of cassette on cell physiology was monitored by determination of optical density, glucose consumption and acetate production. We found that the antisense method partially reduced mRNA levels of the target genes, lowered the concentration of acetate in culture media and increased growth rate and final cell density in antisense-regulated strain. This strategy could provide us with a useful, inexpensive and practical tool to achieve a large-scale protein production system

Cloning and expression of coxsakievirus B3 viral protein-1 in E. coli

Viral protein-1 [VP1] is a major capsid protein of Coxsakievirus B3 [CVB3] that plays an important role in directing viruses towards permissive cells and acts as a main antigenic site of the virus in eliciting of host immune response, hence it seems VP1 can be considered as a vaccine candidate against CVB3 infection. In this study, cDNA of VP1 was prepared, cloned into pET expression vector and the recombinant protein [VP1] was over expressed in E. coli. The viruses were grown in suspension cultures of Vero cells with an input virus multiplicity of 10-50 plaque-forming units/cell. After observing complete cytopathic effect, the total RNA [cells and virus] was prepared for RT-PCR and by using specific primers, VP1 cDNA was amplified and ligated into pET vectors [32 a and 28 a]. The recombinant vector was transferred into competent E. coli [BL-21] and after selection of proper colony, which carried correct cDNA within the vector; cells were cultured and induced with isopropyl B-D-thiogalactopyranoside, in order to express protein [VP1]. The cultures were tested for presence of VP1 by SDS-PAGE and Western-Blotting analysis. Molecular techniques such as PCR which showed exact defined size of the VP1 [819 bp], restriction digestion and finally immunoblot analysis of over expressed protein; all confirmed the correct cloning and expression of VP1 in this research. In this research, full length of VP1 as major capsid protein of CVB3 was over expressed in E. coli which, can be used for further studies, including neutralizing antibody production against CVB3

Cloning and expression of Protease 2 from Coxsakievirus B3

Protease 2A [2Apro] of coxsackievirus B3 [CVB3] plays a major role in viral replication. In case of infection, viral proteins are being synthesized from viral mRNA using host biosynthesis machinery. 2Apro of virus, after being synthesized, exhibits two critical functions, cleavage of viral proteins and breaking eukaryotic initiation factor 4G. The enzyme plays an essential role in viral replication and cellular damage. To understand pathogenicity of infection and also developing potent and selective inhibitors against picornavirus infection, it is necessary to prepare pure 2Apro enzyme. cDNA of 2Apro was synthesized using in vitro infection of permissive host through reverse transcription process and was cloned in pET22b[+]. Since 2Apro is a toxic product, its expression will act on host before induction and damages the cells. For this reason, different hosts were checked and finally BLR[DE3]pLysS, which carries an extra-plasmid for lysozyme expression, that minimizes unwanted target protein products [leakage] was selected. By employing such expression system we could minimize the unwanted expression of 2Apro. Though it is not possible to avoide it, but seems negligible. Hence, this system is useful for expression of toxic proteins in sensitive hosts in order to prevent bacterial damage. The product was confirmed by SDS polyacrylamide gel electrophoresis and immunoblot analysis

Effect of activation and inhibition of cellular PKR on coxsackievirus B3 replication

The ds-RNA activated protein kinase [PKR] is a serine-threonine kinase with MW of 68 KDa. It belongs to a family of kinases that control one of the translational initiation factors, eIF2. PKR is produced at high level in response to viral infection. This protein by phosphorylating eIF2 inhibits cellular protein synthesis. In this study, the effect of gamma interferon [IFN- gamma], an activator, and 2-aminopurine [2AP], an inhibitor of PKR production on coxsackievirus B3 [CVB3] replication were studied. After addition of IFN- gamma and 2AP to Vero and HeLa cultures, cells were infected with CVB3. After 48 h and appearance of cytopathic effect [CPE], cells were collected and viral replication was assessed by standard method. For molecular detection of PKR, RT-PCR technique was used. The data show that interferon inhibited [or lowered] and 2AP promoted viral replication, though in all samples presence of PKR-mRNA could be detected. It seems that PKR plays a key role in CVB3 replication. Therefore, PKR can be considered as a promising and considerable target for designing small molecules and drugs against CVB3