New Strategy for Solubilization and Refolding of Recombinant Human Interferon α2b Inclusion Bodies from E. coli Gene Overexpression System

2018 ◽  
Vol 9 (2) ◽  
pp. 87-93
Author(s):  
Choe Yon ◽  
Pak Yong-Ju ◽  
Yun Song-Jin
Keyword(s):  
Inclusion Bodies ◽  
Human Interferon ◽  
Gene Overexpression ◽  
E Coli ◽  
New Strategy ◽  
Overexpression System ◽  
Interferon Α2b

Preparative method for obtaining recombinant human interferon α2b from inclusion bodies of Escherichia coli

2016 ◽  
Vol 42 (6) ◽  
pp. 631-637 ◽  
Author(s):  
V. P. Romanov ◽  
T. I. Kostromina ◽  
A. I. Miroshnikov ◽  
S. A. Feofanov
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Preparative Method ◽  
Human Interferon ◽  
Interferon Α2b

scholarly journals Comparison of expression systems for the production of human interferon-α2b

2010 ◽  
Vol 5 (4) ◽  
pp. 446-455 ◽  
Author(s):  
Hamid Rajabi Memari ◽  
Ramakrishnan Ramanan ◽  
Arbakariya Ariff
Keyword(s):  
Signal Sequence ◽  
Cauliflower Mosaic Virus ◽  
Human Interferon ◽  
Expression Systems ◽  
E Coli ◽  
Expression Platform ◽  
Yield Quality ◽  
Interferon Α2b ◽  
Selection Of

AbstractThe production of human interferon alpha2b (IFN-α2b) in two expression systems, tobacco (Nicotiana tabaccum) and Escherichia coli, was compared in various aspects such as safety, yield, quality of product and productivity. In the E. coli system, IFN-α2b was expressed under a pelB signal sequence and a T7lac promoter in a pET 26b(+) vector. The same gene was also cloned in expression plant vector (pCAMBIA1304) between cauliflower mosaic virus promoter (CaMV35S) and poly A termination region (Nos) and expressed in transgenic tobacco plants. The expression of protein in both systems was confirmed by western immunoblotting and the quantity of the protein was determined by immunoassay. The amount of periplasmic expression in E. coli was 60 µg/L of culture, while the amount of nuclear expression in the plant was 4.46 µg/kg of fresh leaves. The result of this study demonstrated that IFN-α2b was successfully expressed in periplasm of bacterial and plant systems. The limitations on the production of IFN-α2b by both systems are addressed and discussed to form the basis for the selection of the appropriate expression platform.

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 Optimization of Human Interferon α2b Soluble Protein Overproduction and Primary Recovery of Its Inclusion Bodies

2011 ◽  
Vol 5 (1) ◽  
pp. 27-32 ◽  
Author(s):  
RATIH ASMANA NINGRUM ◽  
DEBBIE SOFIE RETNONINGRUM ◽  
YEYET CAHYATI ◽  
HENI RACHMAWATI
Keyword(s):  
Soluble Protein ◽  
Inclusion Bodies ◽  
Human Interferon ◽  
Protein Overproduction ◽  
Interferon Α2b

Optimization of the primary recovery of human interferon α2b from Escherichia coli inclusion bodies

2006 ◽  
Vol 45 (1) ◽  
pp. 226-234 ◽  
Author(s):  
C.A. Valente ◽  
G.A. Monteiro ◽  
J.M.S. Cabral ◽  
M. Fevereiro ◽  
D.M.F. Prazeres
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Human Interferon ◽  
Interferon Α2b

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.

The role of lac operon and lac repressor in the induction using lactose for the expression of periplasmic human interferon-α2b by Escherichia coli

2011 ◽  
Vol 62 (4) ◽  
pp. 1427-1435 ◽  
Author(s):  
Joo Shun Tan ◽  
Ramakrishnan Nagasundara Ramanan ◽  
Tau Chuan Ling ◽  
Shuhaimi Mustafa ◽  
Arbakariya B. Ariff
Keyword(s):  
Escherichia Coli ◽  
Lac Repressor ◽  
Human Interferon ◽  
Lac Operon ◽  
Interferon Α2b

scholarly journals On-column Refolding of an Insoluble His6-tagged Recombinant EC-SOD Overexpressed in Escherichia coli

2005 ◽  
Vol 37 (4) ◽  
pp. 265-269 ◽  
Author(s):  
Xi-Qiang Zhu ◽  
Su-Xia Li ◽  
Hua-Jun He ◽  
Qin-Sheng Yuan
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Mass Ratio ◽  
Chain Reaction ◽  
Expression Plasmid ◽  
Protein Subunits ◽  
E Coli ◽  
Metal Affinity ◽  
Polymerase Chain ◽  
Escherichia Coli Expression

Abstract The EC-SOD cDNA was cloned by polymerase chain reaction (PCR) and inserted into the Escherichia coli expression plasmid pET-28a(+) and transformed into E. coli BL21(DE3). The corresponding protein that was overexpressed as a recombinant His6-tagged EC-SOD was present in the form of inactive inclusion bodies. This structure was first solubilized under denaturant conditions (8.0 M urea). Then, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione (GSH) and oxidized glutathione (GSSG). The mass ratio of GSH to GSSG was 4:1. The purified enzyme was active, showing that at least part of the protein was properly refolded. The protein was made concentrated by ultrafiltration, and then isolated using Sephacryl S-200 HR. There were two protein peaks in the A280 profile. Based on the results of electrophoresis, we concluded that the two fractions were formed by protein subunits of the same mass, and in the fraction where the molecular weight was higher, the dimer was formed through the disulfide bond between subunits. Activities were detected in the two fractions, but the activity of the dimer was much higher than that of the single monomer. The special activities of the two fractions were found to be 3475 U/mg protein and 510 U/mg protein, respectively.

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