scholarly journals Solubilization of Human Interferon β-1b Inclusion Body Proteins by Organic Solvents

2020 ◽  
Vol 10 (2) ◽  
pp. 233-238
Author(s):  
Samira Nekoufar ◽  
Ahmad Fazeli ◽  
Mohammad Reza Fazeli
Keyword(s):  
Inclusion Body ◽  
Organic Solvents ◽  
Inclusion Bodies ◽  
Research Area ◽  
Human Interferon ◽  
Interferon Β ◽  
Buffer Solution ◽  
Recombinant Interferon ◽  
E Coli ◽  
High Protein Concentration

Purposes: Solubilization of inclusion bodies expressed in E. coli is a critical step during manufacturing of recombinant proteins expressed as inclusion bodies. So far, various methods have been used for solubilization and purification of inclusion body proteins to obtain active proteins with high purity and yield. The aim of this study was to examine the benefit of organic solvents such as alcohols in solubilization of recombinant interferon β-1b inclusion bodies. Methods: Effect of important parameters inclusion pH, concentration and type of denaturant and concentration of alcoholic solvents were optimized to formulate a suitable solubilization buffer and investigate their effect on solubilization of interferon β-1b inclusion bodies. Results: Our findings showed the acidic pH in the range of 2-3 is more suitable than alkaline pH >12 for solubilization and achieving higher content of interferon β-1beta and pure recombinant protein. We have also demonstrated that 1% SDS acts better than 2M urea to solubilize Inclusion body proteins of interferon β-1b at pH of 2-3. The interferon concentration was 2.35 mg per 100 mg IB when we used 40% (v/v) 1-propanol and 20% (v/v) 2-butanol into the buffer solution as well. Conclusion: The optimized method provides gentile condition for solubilization of inclusion body at high protein concentration and purity with a degree of retention of native secondary structure which makes this method valuable to be used in production and research area.

scholarly journals Production of Human Interferon β by Recombinant E. coli Using the Codon Optimized Gene

KSBB Journal ◽  
2017 ◽  
Vol 32 (1) ◽  
pp. 16-21
Author(s):  
Jong-Seok Kim ◽  
Seung-Won Jang ◽  
Jae-Bum Park ◽  
Deok-Ho Kwon ◽  
Young-Jun Chang ◽  
...  
Keyword(s):  
Human Interferon ◽  
Interferon Β ◽  
E Coli

scholarly journals Inclusion Body Bead Size in E. coli Controlled by Physiological Feeding

2018 ◽  
Vol 6 (4) ◽  
pp. 116 ◽  
Author(s):  
Julian Kopp ◽  
Christoph Slouka ◽  
Daniel Strohmer ◽  
Julian Kager ◽  
Oliver Spadiut ◽  
...  
Keyword(s):  
Body Size ◽  
Inclusion Body ◽  
Uptake Rate ◽  
Inclusion Bodies ◽  
Downstream Processing ◽  
Sugar Uptake ◽  
E Coli ◽  
Model Based ◽  
Bead Size

The Gram-negative bacterium E. coli is the host of choice for producing a multitude of recombinant proteins relevant in the pharmaceutical industry. Generally, cultivation is easy, media are cheap, and a high product titer can be obtained. However, harsh induction procedures combined with the usage of IPTG (isopropyl β-d-1 thiogalactopyranoside) as an inducer are often believed to cause stress reactions, leading to intracellular protein aggregates, which are so known as so-called inclusion bodies (IBs). Downstream applications in bacterial processes cause the bottleneck in overall process performance, as bacteria lack many post-translational modifications, resulting in time and cost-intensive approaches. Especially purification of inclusion bodies is notoriously known for its long processing times and low yields. In this contribution, we present screening strategies for determination of inclusion body bead size in an E. coli-based bioprocess producing exclusively inclusion bodies. Size can be seen as a critical quality attribute (CQA), as changes in inclusion body behavior have a major effect on subsequent downstream processing. A model-based approach was used, aiming to trigger a distinct inclusion body size: Physiological feeding control, using qs,C as a critical process parameter, has a high impact on inclusion body size and could be modelled using a hyperbolic saturation mechanism calculated in form of a cumulated substrate uptake rate. Within this model, the sugar uptake rate of the cells, in the form of the cumulated sugar uptake-value, was simulated and considered being a key performance indicator for determination of the desired size. We want to highlight that the usage of the mentioned screening strategy in combination with a model-based approach will allow tuning of the process towards a certain inclusion body size using a qs based control only. Optimized inclusion body size at the time-point of harvest should stabilize downstream processing and, therefore, increase the overall time-space yield. Furthermore, production of distinct inclusion body size may be interesting for application as a biocatalyst and nanoparticulate material.

scholarly journals Downstream processing of recombinant human insulin and its analogues production from E. coli inclusion bodies

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Yin Yin Siew ◽  
Wei Zhang
Keyword(s):  
Inclusion Body ◽  
Human Insulin ◽  
Inclusion Bodies ◽  
Downstream Processing ◽  
Insulin Analogues ◽  
World Health ◽  
Recombinant Human Insulin ◽  
E Coli ◽  
Recombinant Insulin ◽  
Health Organization

AbstractThe Global Diabetes Compact was launched by the World Health Organization in April 2021 with one of its important goals to increase the accessibility and affordability of life-saving medicine—insulin. The rising prevalence of diabetes worldwide is bound to escalate the demand for recombinant insulin therapeutics, and currently, the majority of recombinant insulin therapeutics are produced from E. coli inclusion bodies. Here, a comprehensive review of downstream processing of recombinant human insulin/analogue production from E. coli inclusion bodies is presented. All the critical aspects of downstream processing, starting from proinsulin recovery from inclusion bodies, inclusion body washing, inclusion body solubilization and oxidative sulfitolysis, cyanogen bromide cleavage, buffer exchange, purification by chromatography, pH precipitation and zinc crystallization methods, proinsulin refolding, enzymatic cleavage, and formulation, are explained in this review. Pertinent examples are summarized and the practical aspects of integrating every procedure into a multimodal purification scheme are critically discussed. In the face of increasing global demand for insulin product, there is a pressing need to develop a more efficient and economical production process. The information presented would be insightful to all the manufacturers and stakeholders for the production of human insulins, insulin analogues or biosimilars, as they strive to make further progresses in therapeutic recombinant insulin development and production.

Optimization of medium composition to increase the expression of recombinant human interferon-β using the Plackett–Burman and central composite design in E. coli SE1

3 Biotech ◽  
2021 ◽  
Vol 11 (5) ◽  
Author(s):  
Dharam Pal ◽  
Gopal Patel ◽  
Prakashkumar Dobariya ◽  
Shivraj Hariram Nile ◽  
Abhay H. Pande ◽  
...  
Keyword(s):  
Central Composite Design ◽  
Medium Composition ◽  
Human Interferon ◽  
Interferon Β ◽  
E Coli ◽  
Central Composite

Characterization of E. coli-Derived Recombinant Human Interferon-β as Compared with Fibroblast Human Interferon-β

1987 ◽  
Vol 101 (5) ◽  
pp. 1199-1208 ◽  
Author(s):  
Jun UTSUMI ◽  
Shojiro YAMAZAKI ◽  
Kazuo HOSOI ◽  
Shigenobu KIMURA ◽  
Keizo HANADA ◽  
...  
Keyword(s):  
Human Interferon ◽  
Interferon Β ◽  
E Coli

Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

1987 ◽  
Vol 45 ◽  
pp. 924-925
Author(s):  
F. A. Durum ◽  
R. G. Goldman ◽  
T. J. Bolling ◽  
M. F. Miller
Keyword(s):  
Inclusion Bodies ◽  
Large Scale ◽  
Recombinant Dna ◽  
Test System ◽  
Cell Protein ◽  
Gram Negative Bacteria ◽  
Cytoplasmic Inclusions ◽  
Isolation And Purification ◽  
E Coli ◽  
Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

Suitable Signal Peptides for Secretory Production of Recombinant Granulocyte Colony Stimulating Factor in Escherichia coli

2020 ◽  
Vol 14 (4) ◽  
pp. 269-282
Author(s):  
Sadra S. Tehrani ◽  
Golnaz Goodarzi ◽  
Mohsen Naghizadeh ◽  
Seyyed H. Khatami ◽  
Ahmad Movahedpour ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Clinical Aspects ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
E Coli ◽  
Secretory Production ◽  
Stimulating Factor

Background: Granulocyte colony-stimulating factor (G-CSF) expressed in engineered Escherichia coli (E. coli) as a recombinant protein is utilized as an adjunct to chemotherapy for improving neutropenia. Recombinant proteins overexpression may lead to the creation of inclusion bodies whose recovery is a tedious and costly process. To overcome the problem of inclusion bodies, secretory production might be used. To achieve a mature secretory protein product, suitable signal peptide (SP) selection is a vital step. Objective: In the present study, we aimed at in silico evaluation of proper SPs for secretory production of recombinant G-CSF in E. coli. Methods: Signal peptide website and UniProt were used to collect the SPs and G-CSF sequences. Then, SignalP were utilized in order to predict the SPs and location of their cleavage site. Physicochemical features and solubility were investigated by ProtParam and Protein-sol tools. Fusion proteins sub-cellular localization was predicted by ProtCompB. Results: LPP, ELBP, TSH, HST3, ELBH, AIDA and PET were excluded according to SignalP. The highest aliphatic index belonged to OMPC, TORT and THIB and PPA. Also, the highest GRAVY belonged to OMPC, ELAP, TORT, BLAT, THIB, and PSPE. Furthermore, G-CSF fused with all SPs were predicted as soluble fusion proteins except three SPs. Finally, we found OMPT, OMPF, PHOE, LAMB, SAT, and OMPP can translocate G-CSF into extracellular space. Conclusion: Six SPs were suitable for translocating G-CSF into the extracellular media. Although growing data indicate that the bioinformatics approaches can improve the precision and accuracy of studies, further experimental investigations and recent patents explaining several inventions associated to the clinical aspects of SPs for secretory production of recombinant GCSF in E. coli are required for final validation.

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