scholarly journals Construction of an Artificial Cell Capable of Protein Expression at Low Temperatures Using a Cell Extract Derived from Pseudomonas fluorescens

Processes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 212
Author(s):  
Mana Fukumoto ◽  
Taishi Tonooka
Keyword(s):  
Protein Expression ◽  
Pseudomonas Fluorescens ◽  
Fluorescent Proteins ◽  
Expression System ◽  
Cell Extract ◽  
Bilayer Membrane ◽  
E Coli ◽  
Cold Environments ◽  
Artificial Cell ◽  
A Cell

A liposome-based artificial cell (LBAC) consists of a liposome encapsulating a cell-free protein expression system (CFPES) and protein-encoding DNA. It is surrounded by a lipid bilayer membrane and synthesizes proteins that resemble actual cells. Hence, they have been one of the most studied artificial cells. According to recent studies, they have been able to sense bio-functional molecules by synthesizing fluorescent proteins in response to target molecules. Therefore, they are expected to be used as biosensors. However, previously reported LBACs encapsulated the CFPES derived from Escherichia coli, resulting in the most productive protein expression at 20–40 °C. To broaden the range of their working temperatures to lower temperatures, in this study, we constructed LBACs using a CFPES derived from Pseudomonas fluorescens that grows at a temperature range of 4 °C to 30 °C. We then demonstrated that the constructed LBAC expressed proteins at 8 °C and that, the protein expression capability of the LBAC derived from P. fluorescens was four-fold higher than that derived from E. coli at 8 °C. This study will pave the way for the development of artificial cell-based biosensors that work in cold environments or for the synthesis of heat-labile proteins in LBACs.

scholarly journals Expression of Recombinant Fusion Protein of Newcastle Disease Virus from Escherichia coli Plasmid Clone C-2a by In-vitro Cell-free Protein Expression System

2020 ◽  
Vol 20 ◽  
pp. 04004
Author(s):  
Ahmad Pandu Satria Wiratama ◽  
Aris Haryanto
Keyword(s):  
Protein Expression ◽  
Newcastle Disease ◽  
Recombinant Plasmid ◽  
Expression System ◽  
Free Protein ◽  
F Protein ◽  
E Coli ◽  
Protein Expression System ◽  
Sds Page

Newcastle Disease Virus (NDV) is an infectious disease that infect many kinds of wild and domesticated birds. Infection of NDV become a massive problem for poultry industry around the world especially in Indonesia. Vaccination is an effort to prevent the infection of NDV in poultry. NDV vaccine that used in Indonesia is a conventional life vaccine from LaSota and B1 strains. These type of vaccine is 21%-23% genetically distinct with the virus that spread in the environment. The antibody protection provided by the vaccine is not effective. Therefore, vaccination with new local NDV strain is needed to prevent the NDV infection in Indonesia. The previously study research reported that the local isolate of NDV from Kulon Progo, Indonesia has been isolated. Fusion (F) protein encoding gene that has been inserted into pBT7-N-His expression p lasmid which isolated from clone C-2a of E. coli, then it was expressed by the Cell-free protein expression system. The aim of this study was to confirm whether clone C-2a of E.coli carrying a recombinant plasmid pBT7-N-His-Fusion NDV and to express a recombinant F protein of NDV in-vitro from expression plasmid by cell-free protein expression system. This work started by detection of recombinant plasmid pBT7-N-His-Fusion NDV by DNA plasmid extraction followed by agarose gel electrophoresis. The recombinant F protein was in-vitro expressed by cell-free protein expression kit. The expressed F protein of NDV then was visualized by SDS-PAGE and Westernblott to analyse the expression of NDV recombinant F protein. It confirmed that clone C-2a of E. coli contained plasmid pBT7-N-His (4.001 bp) inserted by recombinant F protein of NDV gene (642 bp). The visualisation of expressed recombinant F protein by SDS-PAGE and Westernblott showed the NDV recombinant F protein was a specific protein fragment with molecular weight of 25,6 kDa..

scholarly journals A new dual prokaryotic (E. coli) and mammalian expression system (pgMAXs)

2020 ◽  
Author(s):  
Manabu Murakami ◽  
Agnieszka M. Murakami ◽  
Kazuyoshi Hirota ◽  
Shirou Itagaki
Keyword(s):  
Topoisomerase Ii ◽  
Fluorescent Proteins ◽  
Expression System ◽  
Simple Expression ◽  
Amino Acid Sequences ◽  
Expression Plasmid ◽  
Iptg Induction ◽  
E Coli ◽  
Mammalian Expression ◽  
Effective Selection

AbstractWe introduce an efficient subcloning and expression plasmid system with two different modes (prokaryotic for expression in Escherichia coli with lac promoter and mammalian modes with cytomegalovirus promoter). The efficient subcloning (DNA insertion) is based upon a DNA topoisomerase II toxin-originated gene for effective selection with isopropyl-β-D-thiogalactoside (IPTG) induction. The new pgMAXs system is manageable size (4452 bp) and has also various types of protein tags (flag, myc, poly-histidine, Human influenza hemagglutinin, strep, and v5) for expression analysis. With pgMAXs system, various types of fluorescent proteins were subcloned and prtein expressions were confirmed. We also tried to identify epitope amino acid sequences for anti-calcium channel β2 antibody, by constructing epitope-library with DNaseI-partial digestion and subcloning into EcoRV site in pgMAXs. The new pgMAXs plasmid system enables highly efficient subcloning, simple expression in E. coli and that it has a simple deletion step of rare 8-nucleotide rare-cutter blunt-end enzymes for mammalian expression plasmid construction. Taken together, the pgMAXs system simplifies prokaryotic and mammalian gene expression analyses.

scholarly journals Codon Preference Optimization Increases Prokaryotic Cystatin C Expression

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Qing Wang ◽  
Cui Mei ◽  
Honghua Zhen ◽  
Jess Zhu
Keyword(s):  
Protein Expression ◽  
Cystatin C ◽  
Production Systems ◽  
Codon Optimization ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
Gc Content ◽  
Optimization Techniques ◽  
E Coli ◽  
Prokaryotic Expression Vector

Gene expression is closely related to optimal vector-host system pairing in many prokaryotes. Redesign of the humancystatin C(cysC) gene using the preferred codons of the prokaryotic system may significantly increasecysCexpression inEscherichia coli(E. coli). Specifically,cysCexpression may be increased by removing unstable sequences and optimizing GC content. According toE. coliexpression system codon preferences, the gene sequence was optimized while the amino acid sequence was maintained. The codon-optimizedcysC(co-cysC) and wild-typecysC(wt-cysC) were expressed by cloning the genes into a pET-30a plasmid, thus transforming the recombinant plasmid intoE. coliBL21. Before and after the optimization process, the prokaryotic expression vector and host bacteria were examined for protein expression and biological activation of CysC. The recombinant proteins in the lysate of the transformed bacteria were purified using Ni2+-NTA resin. Recombinant protein expression increased from 10% to 46% based on total protein expression after codon optimization. Recombinant CysC purity was above 95%. The significant increase incysCexpression inE. coliexpression produced by codon optimization techniques may be applicable to commercial production systems.

scholarly journals In vitro expression of the recombinant fusion protein of Newcastle disease virus from local Indonesian isolates by using a cell-free protein expression system

2020 ◽  
Vol 25 (2) ◽  
pp. 69
Author(s):  
Aris Haryanto ◽  
Hevi Wihadmadyatami ◽  
Nastiti Wijayanti
Keyword(s):  
Protein Expression ◽  
Newcastle Disease Virus ◽  
Restriction Endonuclease ◽  
Newcastle Disease ◽  
Expression System ◽  
Free Protein ◽  
F Protein ◽  
Protein Expression System ◽  
A Cell

The aim of this work was the in vitro expression of the recombinant fusion (F) protein of Newcastle disease virus (NDV).  The pBT7-N-His-Fusion-NDV expression plasmid which carries the recombinant F protein encoding gene from local Indonesian isolates, was prepared and transformed into E. coli BL21 (DE3). To detect bacterial colonies carrying the recombinant plasmid, a restriction endonuclease analysis was performed using the EcoRI restriction endonuclease. These results showed that the pBT-N-His-Fusion-NDV plasmid was successfully isolated with a size of 4.601 bp, and three recombinant plasmids carrying the gene coding for the recombinant F protein of NDV were obtained. Selected recombinant plasmids were then in vitro by using a cell-free protein expression system followed by visualization of the recombinant F protein on a 12% SDS-PAGE gel both by Coomassie Brilliant Blue staining and Western blotting. Recombinant F protein was successfully in vitro expressed by using a cell-free protein expression system as indicated by a specific single protein band with a molecular mass of 25.6 kDa.

scholarly journals Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21

2021 ◽  
Vol 12 ◽  
Author(s):  
Gema Lozano Terol ◽  
Julia Gallego-Jara ◽  
Rosa Alba Sola Martínez ◽  
Adrián Martínez Vivancos ◽  
Manuel Cánovas Díaz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Copy Number ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
E Coli

Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB1′ and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.

scholarly journals Efficient production of immunologically active Shigella invasion plasmid antigens IpaB and IpaH using a cell-free expression system

2021 ◽  
Author(s):  
Neeraj Kapoor ◽  
Esther Ndungo ◽  
Lucy Pill ◽  
Girmay Desalegn ◽  
Aym Berges ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Expression System ◽  
Efficient Production ◽  
Biophysical Characterization ◽  
Antimicrobial Drug Resistance ◽  
E Coli ◽  
Key Points ◽  
Sds Page ◽  
A Cell ◽  
Conventional Cell

Abstract Shigella spp. invade the colonic epithelium and cause bacillary dysentery in humans. Individuals living in areas that lack access to clean water and sanitation are the most affected. Even though infection can be treated with antibiotics, Shigella antimicrobial drug resistance complicates clinical management. Despite decades of effort, there are no licensed vaccines to prevent shigellosis. The highly conserved invasion plasmid antigens (Ipa), which are components of the Shigella type III secretion system, participate in bacterial epithelial cell invasion and have been pursued as vaccine targets. However, expression and purification of these proteins in conventional cell-based systems have been challenging due to solubility issues and extremely low recovery yields. These difficulties have impeded manufacturing and clinical advancement. In this study, we describe a new method to express Ipa proteins using the Xpress+TM cell-free protein synthesis (CFPS) platform. Both IpaB and the C-terminal domain of IpaH1.4 (IpaH-CTD) were efficiently produced with this technology at yields > 200 mg/L. Furthermore, the expression was linearly scaled in a bioreactor under controlled conditions, and proteins were successfully purified using multimode column chromatography to > 95% purity as determined by SDS-PAGE. Biophysical characterization of the cell-free synthetized IpaB and IpaH-CTD using SEC-MALS analysis showed well-defined oligomeric states of the proteins in solution. Functional analysis revealed similar immunoreactivity as compared to antigens purified from E. coli. These results demonstrate the efficiency of CFPS for Shigella protein production; the practicality and scalability of this method will facilitate production of antigens for Shigella vaccine development and immunological analysis. Key points • First report of Shigella IpaB and IpaH produced at high purity and yield using CFPS • CFPS-IpaB and IpaH perform similarly to E. coli–produced proteins in immunoassays • CFPS-IpaB and IpaH react with Shigella-specific human antibodies and are immunogenic in mice. Graphical abstract

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