scholarly journals Expression, Purification, and Characterization of a Sucrose Nonfermenting 1-Related Protein Kinases 2 of Arabidopsis thaliana in E. coli-Based Cell-Free System

2016 ◽  
Vol 2016 ◽  
pp. 1-8
Author(s):  
Xu Zhang ◽  
Shuangxi Zhang ◽  
Jun Wang ◽  
Jing Zi ◽  
Jianhui Wang ◽  
...  
Keyword(s):  
Large Scale ◽  
Biochemical Properties ◽  
Plant Responses ◽  
Scale Production ◽  
Related Protein ◽  
Protein Preparation ◽  
Free System ◽  
Cell Free System ◽  
E Coli

The plant-specific sucrose nonfermenting 1-related protein kinase 2 (SnRK2) family is considered an important regulator of plant responses to abiotic stresses such as drought, cold, salinity, and nutrition deficiency. However, little information is available on how SnRK2s regulate sulfur deprivation responses in Arabidopsis. Large-scale production of SnRK2 kinases in vitro can help to elucidate the biochemical properties and physiological functions of this protein family. However, heterogenous expression of SnRK2s usually leads to inactive proteins. In this study, we expressed a recombinant Arabidopsis SnRK2.1 in a modified E. coli cell-free system, which combined two kinds of extracts allowing for a convenient and affordable protein preparation. The recombinant SnRK2.1 was produced in large-scale and the autophosphorylation activity of purified SnRK2.1 was characterized, allowing for further biochemical and substrate binding analysis in sulfur signaling. The application of this improved E. coli cell-free system provides us a promising and convenient platform to enhance expression of the target proteins economically.

In vitro protein synthesis directed by R17 viral ribonucleic acid. II. On the fate of mRNA in the cell-free system

1969 ◽  
Vol 47 (12) ◽  
pp. 1179-1186 ◽  
Author(s):  
Satomi J. Igarashi
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid Incorporation ◽  
Free System ◽  
Cell Free System ◽  
E Coli ◽  
Acid Incorporation ◽  
Binding Factor ◽  
Vitro Protein

In the crude E. coli B cell-free system, mRNA was hydrolyzed by contaminating nuclease activities before significant polymerization of amino acids took place. Ribosomes appeared to be one of the sources of nuclease. A modified high-salt washing procedure was developed to remove nuclease from ribosomes. RNase-free ribosomes thus obtained appeared to be inactive in poly-U-directed phenylalanine incorporation, unless poly-U binding factor was added to the system. R17 RNA could not direct amino acid incorporation in the presence of RNase-free ribosomes because binding of intact R17 RNA to ribosomes did not take place even in the presence of poly-U binding factor.

scholarly journals Biosynthesis and characterization of silver nanoparticles induced by fungal proteins and its application in different biological activities

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Abdelmageed M. Othman ◽  
Maysa A. Elsayed ◽  
Naser G. Al-Balakocy ◽  
Mohamed M. Hassan ◽  
Ali M. Elshafei
Keyword(s):  
Antimicrobial Activity ◽  
Silver Nanoparticles ◽  
Large Scale ◽  
Biological Activities ◽  
Spherical Shape ◽  
Inhibition Zone ◽  
Scale Production ◽  
E Coli ◽  
Biosynthesized Agnps

Abstract Background The present study aims to apply an efficient eco-friendly and inexpensive process for green synthesis of silver nanoparticles (AgNPs) through the mediation of fungal proteins from Aspergillus fumigatus DSM819, characterization, and its application as antimicrobial finishing agent in textile fabrics against some infectious microorganisms. Results Optimum conditions for AgNP biosynthesis could be achieved by means of using 60% (v/v) of cell-free filtrate (CFF) and 1.5 mM of AgNO3 at pH 10.0 after 90 min. The obtained AgNPs were of spherical shape with 90% of distribution below than 84.4 nm. The biosynthesized AgNPs exerted an antimicrobial activity against the studied pathogenic microorganisms (E. coli, B. mycoides, and C. albicans). In addition, IC50 values against in vitro tumor cell lines were found to be 31.1, 45.4, 40.9, and 33.5 μg/ml for HCT116, A549, MCF7, and PC3, respectively. Even with a very low concentration (0.25%), the treated PET/C fabrics by AgNPs exerted an antimicrobial activity against E. coli, B. mycoides, and C. albicans to give inhibition zone diameter of 15, 15, and 16 mm, respectively. Conclusions The green biosynthesis approach applied in this study is a non-toxic alternative to the traditional chemical and physical methods, and would be appropriate for biological large-scale production and prospective treatments. Graphical abstract

scholarly journals A fractionated cell-free system for analysis of prophase nuclear disassembly.

1986 ◽  
Vol 103 (6) ◽  
pp. 2073-2081 ◽  
Author(s):  
F A Suprynowicz ◽  
L Gerace
Keyword(s):  
Chromatin Condensation ◽  
Chinese Hamster ◽  
Nuclear Lamina ◽  
Biochemical Properties ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Cell Nuclei ◽  
Free System ◽  
Cell Free System

We describe a cell-free system in which a postribosomal supernatant (s140) from metaphase Chinese hamster ovary (CHO) cells induces prophase-like changes in isolated CHO cell nuclei, including chromatin condensation, and nuclear envelope and lamina disassembly. These events are strongly promoted by gamma-S-ATP and an ATP-regenerating system, and do not take place with an s140 derived from G2-phase cells. The metaphase cell s140 also induces disassembly of an isolated nuclear lamina fraction that is depleted of membranes, chromatin, and nuclear pore complexes. Disassembly of the isolated lamina is accompanied by phosphorylation of the major lamina proteins (lamins A, B, and C) to levels characteristic of metaphase cells. Kinetic analysis of lamina depolymerization indicates that cooperativity may be involved in this process. The biochemical properties of in vitro lamina disassembly suggest that the activity that depolymerizes the lamina during mitosis is soluble in metaphase cells, and support the notion that this activity is a lamin protein kinase.

scholarly journals Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1288
Author(s):  
Wendy Dong ◽  
Boris Kantor
Keyword(s):  
Large Scale ◽  
Lentiviral Vector ◽  
Clinical Applications ◽  
Scale Production ◽  
Base Editing ◽  
Epigenome Editing ◽  
Vector Systems ◽  
Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

High-Level Expression of Human β-Defensin-2 Gene with Rare Codons in E. coli Cell-Free System

2006 ◽  
Vol 13 (2) ◽  
pp. 155-161 ◽  
Author(s):  
Haiqin Chen ◽  
Zhinan Xu ◽  
Naizheng Xu ◽  
Peilin Cen
Keyword(s):  
Coli Cell ◽  
High Level Expression ◽  
Free System ◽  
Cell Free System ◽  
E Coli ◽  
Rare Codons ◽  
High Level

scholarly journals Enzyme-catalyzed biodegradation of penicillin fermentation residues by β-lactamase OtLac from Ochrobactrum tritici

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Peng Wang ◽  
Chen Shen ◽  
Qinqin Cong ◽  
Kaili Xu ◽  
Jialin Lu
Keyword(s):  
Large Scale ◽  
In Vitro Study ◽  
Scale Removal ◽  
E Coli ◽  
Penicillin V ◽  
Highly Active ◽  
Steady State Kinetics ◽  
Biodegradation Process ◽  
Km Value

Abstract Background Biodegradation of antibiotics is a promising method for the large-scale removal of antibiotic residues in the environment. However, the enzyme that is involved in the biodegradation process is the key information to be revealed. Results In this study, the beta-lactamase from Ochrobactrumtritici that mediates the biodegradation of penicillin V was identified and characterized. When searching the proteins of Ochrobactrumtritici, the β-lactamase (OtLac) was identified. OtLac consists of 347 amino acids, and predicted isoelectric point is 7.0. It is a class C β-lactamase according to BLAST analysis. The coding gene of OtLac was amplified from the genomic DNA of Ochrobactrumtritici. The OtLac was overexpressed in E. coli BL21 (DE3) and purified with Ni2+ column affinity chromatography. The biodegradation ability of penicillin V by OtLac was identified in an in vitro study and analyzed by HPLC. The optimal temperature for OtLac is 32 ℃ and the optimal pH is 7.0. Steady-state kinetics showed that OtLac was highly active against penicillin V with a Km value of 17.86 μM and a kcat value of 25.28 s−1 respectively. Conclusions OtLac demonstrated biodegradation activity towards penicillin V potassium, indicating that OtLac is expected to degrade penicillin V in the future.

scholarly journals Cloning, expression, and analysis of the group 2 allergen from Dermatophagoides farinae from China

2010 ◽  
Vol 82 (4) ◽  
pp. 941-951 ◽  
Author(s):  
Cui Yu-bao ◽  
Ying Zhou ◽  
Shi Weihong ◽  
Ma Guifang ◽  
Li Yang ◽  
...  
Keyword(s):  
Large Scale ◽  
Alpha Helix ◽  
Random Coil ◽  
Reference Sequence ◽  
Scale Production ◽  
Dermatophagoides Farinae ◽  
E Coli ◽  
Large Scale Production ◽  
Solid Foundation ◽  
Group 2

To obtain the recombinant group 2 allergen product of Dermatophagoides farinae (Der f 2), the Der f 2 gene was synthesized by RT-PCR. The full-length cDNA comprised 441 nucleotides and was 99.3% identical to the reference sequence (GenBank AB195580). The cDNA was bound to vector pET28a to construct plasmid pET28a(+)-Der f 2, which was transformed into E. coli BL21 and induced by IPTG. SDS-PAGE showed a specific band of about 14kDa in the hole cell lysate. s estiated by chroatography, about 3.86 g of the recobinant product as obtained, which conjugated with serum IgE from asthmatic children. The protein had a signal peptide of 17 amino acids. Its secondary structure comprised an alpha helix (19.86%), an extended strand (30.82%), and a random coil (49.32%). The subcellular localization of this allergen was predicted to be at mitochondria. Furthermore, its function was shown to be associated with an MD-2-related lipid-recognition (ML) domain. The results of this study provide a solid foundation for large-scale production of the allergen for clinical diagnosis and treatent of allergic disorders.

An economical method for producing stable-isotope labeled proteins by the E. coli cell-free system

2010 ◽  
Vol 48 (4) ◽  
pp. 193-201 ◽  
Author(s):  
Jun Yokoyama ◽  
Takayoshi Matsuda ◽  
Seizo Koshiba ◽  
Takanori Kigawa
Keyword(s):  
Stable Isotope ◽  
Coli Cell ◽  
Free System ◽  
Economical Method ◽  
Cell Free System ◽  
E Coli
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