A novel technique for the measurement of disruption in high-pressure homogenization: Studies on E. coli containing recombinant inclusion bodies

1991 ◽  
Vol 38 (4) ◽  
pp. 363-370 ◽  
Author(s):  
Anton P. J. Middelberg ◽  
Brian K. O'Neill ◽  
I. David L. Bogle ◽  
Mark A. Snoswell
Keyword(s):  
High Pressure ◽  
Inclusion Bodies ◽  
High Pressure Homogenization ◽  
E Coli ◽  
Novel Technique

Machine learning modelling for the high-pressure homogenization-mediated disruption of recombinant E. coli

2018 ◽  
Vol 71 ◽  
pp. 182-190 ◽  
Author(s):  
Kiran D. Bhilare ◽  
Mahesh D. Patil ◽  
Sujit Tangadpalliwar ◽  
Manoj J. Dev ◽  
Prabha Garg ◽  
...  
Keyword(s):  
Machine Learning ◽  
High Pressure ◽  
High Pressure Homogenization ◽  
E Coli

scholarly journals Selective Release of Recombinant Periplasmic Protein From E. coli Using Continuous Pulsed Electric Field Treatment

2021 ◽  
Vol 8 ◽  
Author(s):  
Felix Schottroff ◽  
Jens Kastenhofer ◽  
Oliver Spadiut ◽  
Henry Jaeger ◽  
David J. Wurm
Keyword(s):  
High Pressure ◽  
Recombinant Protein ◽  
Electric Field ◽  
Host Cell ◽  
Energy Input ◽  
Periplasmic Protein ◽  
High Pressure Homogenization ◽  
E Coli ◽  
Product Release ◽  
Cell Disintegration

To date, high-pressure homogenization is the standard method for cell disintegration before the extraction of cytosolic and periplasmic protein from E. coli. Its main drawback, however, is low selectivity and a resulting high load of host cell impurities. Pulsed electric field (PEF) treatment may be used for selective permeabilization of the outer membrane. PEF is a process which is able to generate pores within cell membranes, the so-called electroporation. It can be readily applied to the culture broth in continuous mode, no additional chemicals are needed, heat generation is relatively low, and it is already implemented at industrial scale in the food sector. Yet, studies about PEF-assisted extraction of recombinant protein from bacteria are scarce. In the present study, continuous electroporation was employed to selectively extract recombinant Protein A from the periplasm of E. coli. For this purpose, a specifically designed flow-through PEF treatment chamber was deployed, operated at 1.5 kg/h, using rectangular pulses of 3 μs at specific energy input levels between 10.3 and 241.9 kJ/kg. Energy input was controlled by variation of the electric field strength (28.4–44.8 kV/cm) and pulse repetition frequency (50–1,000 Hz). The effects of the process parameters on cell viability, product release, and host cell protein (HCP), DNA, as well as endotoxin (ET) loads were investigated. It was found that a maximum product release of 89% was achieved with increasing energy input levels. Cell death also gradually increased, with a maximum inactivation of -0.9 log at 241.9 kJ/kg. The conditions resulting in high release efficiencies while keeping impurities low were electric field strengths ≤ 30 kV/cm and frequencies ≥ 825 Hz. In comparison with high-pressure homogenization, PEF treatment resulted in 40% less HCP load, 96% less DNA load, and 43% less ET load. Therefore, PEF treatment can be an efficient alternative to the cell disintegration processes commonly used in downstream processing.

Recombinant murine growth hormone from E. coli inclusion bodies: Expression, high-pressure solubilization and refolding, and characterization of activity and structure

2010 ◽  
Vol 26 (3) ◽  
pp. 743-749 ◽  
Author(s):  
Amber Haynes Fradkin ◽  
Carl S. Boand ◽  
Stephen P. Eisenberg ◽  
Mary S. Rosendahl ◽  
Theodore W. Randolph
Keyword(s):  
Growth Hormone ◽  
High Pressure ◽  
Inclusion Bodies ◽  
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.

High-Pressure Homogenization Alters Physicochemical Properties and In Vitro Digestibility of Chinese Yam (Dioscorea Opposita Thunb.) Starch

2018 ◽  
Vol 18 (1) ◽  
pp. 10-15
Author(s):  
Wang Yi-Wei ◽  
He Yong-Zhao ◽  
An Feng-Ping ◽  
Huang Qun ◽  
Zeng Feng ◽  
...  
Keyword(s):  
High Pressure ◽  
Physicochemical Properties ◽  
Starch Content ◽  
In Vitro Digestibility ◽  
High Pressure Homogenization ◽  
Starch Granules ◽  
Cross Polarization ◽  
Chinese Yam ◽  
Crystal Type

In this study, Chinese yam starch-water suspension (8%) were subjected to high-pressure homogenization (HPH) at 100 MPa for increasing cycle numbers, and its effect of on the physicochemical properties of the starch was investigated. Results of the polarizing microscope observations showed that the starch granules were disrupted (i.e. greater breakdown value) after HPH treatment, followed by a decrease in cross polarization. After three HPH cycles, the crystallinity of starch decreased, while the crystal type remained unaltered. Meanwhile, the contents of rapidly digestible starch and slowly digestible starch were increased. On the contrary, resistant starch content was decreased. Our results indicate that HPH treatment resulted in reduction of starch crystallinity and increase of starch digestibility.

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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