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

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