Optimizing conditions for production of high levels of soluble recombinant human growth hormone using Taguchi method

Savari, Marzieh; Zarkesh-Esfahani, Hamid; Edalati, Masoud; Biria, Davoud

doi:10.1016/j.pep.2015.06.006

Cited by 18 publications

(13 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Hezarjaribi et al [33] for creating the highest protein production by Saccharomyces cerevisiae Using an Optimized Culture Medium Composition use the Taguchi method by Qualitek-4 software as a fractional factorial statistical method and they reported that Due to high obtained protein production in dry cell biomass of S. cerevisiae PTCC5269, there is a good potential to commercialization of this procedure. Savari et al [34] to achieve higher levels of soluble form of recombinant human growth hormone in E. coli used Taguchi method and 32 experiments were designed using Minitab 16 software with evaluation of several factors such as the effects of different temperatures, IPTG concentrations, induction periods, E. coli strains, glycerol and ethanol. Taguchi method allows accurate optimization of a process by performing the minimum numbers of experiments possible.…”

Section: Introductionmentioning

confidence: 99%

Taguchi Experimental Design for Optimization of Recombinant Human Growth Hormone Production in CHO Cell Lines and Comparing its Biological Activity with Prokaryotic Growth Hormone

Aghili

Zarkesh-Esfahani

2017

Drug Res (Stuttg)

View full text Add to dashboard Cite

Growth hormone deficiency results in growth retardation in children and the GH deficiency syndrome in adults and they need to receive recombinant-GH in order to rectify the GH deficiency symptoms. Mammalian cells have become the favorite system for production of recombinant proteins for clinical application compared to prokaryotic systems because of their capability for appropriate protein folding, assembly, post-translational modification and proper signal. However, production level in mammalian cells is generally low compared to prokaryotic hosts. Taguchi has established orthogonal arrays to describe a large number of experimental situations mainly to reduce experimental errors and to enhance the efficiency and reproducibility of laboratory experiments.In the present study, rhGH was produced in CHO cells and production of rhGH was assessed using Dot blotting, western blotting and Elisa assay. For optimization of rhGH production in CHO cells using Taguchi method An M16 orthogonal experimental design was used to investigate four different culture components. The biological activity of rhGH was assessed using LHRE-TK-Luciferase reporter gene system in HEK-293 and compared to the biological activity of prokaryotic rhGH.A maximal productivity of rhGH was reached in the conditions of 1%DMSO, 1%glycerol, 25 µM ZnSO and 0 mM NaBu. Our findings indicate that control of culture conditions such as the addition of chemical components helps to develop an efficient large-scale and industrial process for the production of rhGH in CHO cells. Results of bioassay indicated that rhGH produced by CHO cells is able to induce GH-mediated intracellular cell signaling and showed higher bioactivity when compared to prokaryotic GH at the same concentrations.

show abstract

Section: Introductionmentioning

confidence: 99%

Taguchi Experimental Design for Optimization of Recombinant Human Growth Hormone Production in CHO Cell Lines and Comparing its Biological Activity with Prokaryotic Growth Hormone

Aghili

Zarkesh-Esfahani

2017

Drug Res (Stuttg)

View full text Add to dashboard Cite

show abstract

“…It has been widely reported that induction parameters can affect protein expression level in shake-flask culture (Azaman et al 2010;Larentis et al 2011;Savari et al 2015;Gholami Tilko et al 2016). In the present study for the first time, the effect of post-induction time on β-NGF production level was investigated in a 5-l bioreactor.…”

Section: Resultsmentioning

confidence: 99%

“…3 361 entis et al 2011;Papaneophytou and Kontopidis 2012;Papaneophytou et al 2013;Savari et al 2015;Gholami Tilko et al 2016). For example, in a survey recently conducted by our laboratory optimization of recombinant β-NGF production in shake-flask culture was done (Gholami Tilko et al 2016).…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, in the case of expression of complex heterologous proteins, usually a decrease in the temperature during protein synthesis especially after IPTG induction and, consequently, slower growth rate of bacteria favor the production of the active protein form. It is also important to note that in the literature different post induction temperatures have been reported for overproduction of different heterologous proteins in the bioreactor scale or shake-flask cultures (Azaman et al 2010;Papaneophytou and Kontopidis 2012;Morowvat et al 2015;Savari et al 2015;Su et al 2015;Gholami Tilko et al 2016). Therefore, the evaluation of the best post induction temperature for β-NGF production on bioreactor scale was one of the goals of this study.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Improved Production of Recombinant Human β-NGF in Escherichia coli – a Bioreactor Scale Study

Hajihassan

Tilko

Sadat

2018

Polish Journal of Microbiology

View full text Add to dashboard Cite

A b s t r a c tHuman nerve growth factor β (β-NGF) is considered a major therapeutic agent for treatment of neurodegenerative diseases. We have previously reported the optimized conditions for β-NGF overproduction in Escherichia coli in a shake-flask culture. In this study the optimal %DO (dissolved oxygen) and post induction temperature values for improved production of β-NGF were found in the bioreactor scale using response surface methodology (RSM) as the most common statistical method. Also, for further enhancement of the yield, different post-induction periods of time were selected for testing. In all experiments, the productivity level and bacterial cell growth were evaluated by western blotting technique and monitoring of absorbance at 600 nm, respectively. Our results indicated that %DO, the post-induction time and temperature have significant effects on the production of β-NGF. After 2 hours of induction, the low post induction temperature of 32°C and 20% DO were used to increase the production of β-NGF in a 5-l bioreactor. Another important result obtained in this study was that the improved β-NGF production was not achieved at highest dry cell weigh or highest cell growth. These results are definitely of importance for industrial β-NGF production.K e y w o r d s: bioreactor, RSM, β-NGF over production, E. coli Hajihassan Z. et al.

show abstract

“…The DoE method has been broadly employed for process optimization in the engineering and manufacturing industry, whereas it is not widely used in the biomedical field. Still, there have been some reports on the optimization of protein expression or assay development [63,64,65,66]. This method provides a straightforward tool to rationally analyze factors affecting BoNT substrate cleavage with limited experimental efforts.…”

Section: Introductionmentioning

confidence: 99%

Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A–F Employing Taguchi Design-of-Experiments

Berg

Stern

Weisemann

et al. 2019

Toxins

View full text Add to dashboard Cite

The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins’ light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A–F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A–F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn2+, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn2+, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays.

show abstract

Optimizing conditions for production of high levels of soluble recombinant human growth hormone using Taguchi method

Cited by 18 publications

References 28 publications

Taguchi Experimental Design for Optimization of Recombinant Human Growth Hormone Production in CHO Cell Lines and Comparing its Biological Activity with Prokaryotic Growth Hormone

Taguchi Experimental Design for Optimization of Recombinant Human Growth Hormone Production in CHO Cell Lines and Comparing its Biological Activity with Prokaryotic Growth Hormone

Improved Production of Recombinant Human β-NGF in Escherichia coli – a Bioreactor Scale Study

Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A–F Employing Taguchi Design-of-Experiments

Contact Info

Product

Resources

About