scholarly journals A Spider Toxin Exemplifies the Promises and Pitfalls of Cell-Free Protein Production for Venom Biodiscovery

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 575
Author(s):  
Tim Lüddecke ◽  
Anne Paas ◽  
Lea Talmann ◽  
Kim N. Kirchhoff ◽  
Björn M. von Reumont ◽  
...  
Keyword(s):  
Protein Production ◽  
Bioactive Peptides ◽  
Scale Production ◽  
Expression Systems ◽  
Alternative Strategies ◽  
Limited Size ◽  
Free Protein ◽  
Sds Page ◽  
Rapid Laboratory

Arthropod venoms offer a promising resource for the discovery of novel bioactive peptides and proteins, but the limited size of most species translates into minuscule venom yields. Bioactivity studies based on traditional fractionation are therefore challenging, so alternative strategies are needed. Cell-free synthesis based on synthetic gene fragments is one of the most promising emerging technologies, theoretically allowing the rapid, laboratory-scale production of specific venom components, but this approach has yet to be applied in venom biodiscovery. Here, we tested the ability of three commercially available cell-free protein expression systems to produce venom components from small arthropods, using U2-sicaritoxin-Sdo1a from the six-eyed sand spider Hexophtalma dolichocephala as a case study. We found that only one of the systems was able to produce an active product in low amounts, as demonstrated by SDS-PAGE, mass spectrometry, and bioactivity screening on murine neuroblasts. We discuss our findings in relation to the promises and limitations of cell-free synthesis for venom biodiscovery programs in smaller invertebrates.

scholarly journals Predictive Approaches to Guide the Expression of Recombinant Vaccine Targets in Escherichia Coli: A Case Study Presentation Utilising Absynth Biologics Ltd Proprietary C.Difficile Vaccine Antigens

2021 ◽  
Author(s):  
HIRRA HUSSAIN ◽  
Edward A McKenzie ◽  
Andrew M Robinson ◽  
Neill A Gingles ◽  
Fiona Marston ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Fusion Proteins ◽  
Recombinant Protein Production ◽  
Inclusion Bodies ◽  
Protein Production ◽  
Expression Systems ◽  
Predictive Approaches ◽  
Bacterial Expression Systems

Abstract Background: Bacterial expression systems remain a widely used host for recombinant protein production. However, overexpression of recombinant target proteins in bacterial systems such as Escherichia coli can result in poor solubility and the formation of insoluble aggregates, termed inclusion bodies. As a consequence, different and numerous strategies or alternative engineering approaches have been employed to increase recombinant protein production. In this case study, we present the strategies used to increase the recombinant production and solubility of ‘difficult-to-express’ bacterial antigens, termed Ant2 and Ant3, from Absynth Biologics Ltd’s Clostridium difficile vaccine programme. Results: Single recombinant antigens (Ant2 and Ant3) and fusion proteins (Ant2-3 and Ant3-2) formed insoluble aggregates (inclusion bodies) when overexpressed in BL21 CodonPlus (DE3) cells. Further, proteolytic cleavage of Ant2-3 was observed, potentially due to the presence of a large un-structured loop between the protein boundaries. Optimisation of culture conditions such as varying the induction temperature and addition of heat-shock inducer benzyl alcohol to the growth media had no significant effect on the processing and protein production pattern for all four antigen molecules. Changes to the construct design to include N-terminal solubility tags (Thioredoxin and N utilisation substance protein A) did not improve solubility. Screening of different buffer/additives to improve stability showed that the addition of 1-15mM dithiothreitol (DTT) alone improved the stability of both Ant2 and Ant3. Structural models were generated for Ant2 and Ant3 and solubility-based prediction tools were employed to determine the role of charge and hydrophobicity on protein production. The results showed that both Ant2 and Ant3 contained unfavorable features associated with poor solubility. A large non-polar region was detected on the surface of Ant2 structures, whereas, positively charged regions were observed for Ant3.Conclusions: Commonly used strategies to enhance recombinant protein production in bacterial systems did not act to increase production of model ‘difficult-to-express’ antigens, Ant2 and Ant3 and their fusion proteins. Sequence and structural analysis of antigens identified unfavorable features that potentially result in the increased tendency of these antigens to aggregate and/or lead to improper processing. We present a guide of strategies and predictive approaches that aim to guide the construct design, prior to expression studies, to define and engineer sequences/structures that could lead to increased expression of single and potentially multi-domain (or fusion) antigens in bacterial expression systems.

scholarly journals Predictive approaches to guide the expression of recombinant vaccine targets in Escherichia coli: a case study presentation utilising Absynth Biologics Ltd. proprietary Clostridium difficile vaccine antigens

2021 ◽  
Author(s):  
Hirra Hussain ◽  
Edward A McKenzie ◽  
Andrew M Robinson ◽  
Neill A Gingles ◽  
Fiona Marston ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Clostridium Difficile ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Expression Systems ◽  
Predictive Approaches ◽  
Bacterial Expression Systems

AbstractBacterial expression systems remain a widely used host for recombinant protein production. However, overexpression of recombinant target proteins in bacterial systems such as Escherichia coli can result in poor solubility and the formation of insoluble aggregates. As a consequence, numerous strategies or alternative engineering approaches have been employed to increase recombinant protein production. In this case study, we present the strategies used to increase the recombinant production and solubility of ‘difficult-to-express’ bacterial antigens, termed Ant2 and Ant3, from Absynth Biologics Ltd.’s Clostridium difficile vaccine programme. Single recombinant antigens (Ant2 and Ant3) and fusion proteins (Ant2-3 and Ant3-2) formed insoluble aggregates (inclusion bodies) when overexpressed in bacterial cells. Further, proteolytic cleavage of Ant2-3 was observed. Optimisation of culture conditions and changes to the construct design to include N-terminal solubility tags did not improve antigen solubility. However, screening of different buffer/additives showed that the addition of 1–15 mM dithiothreitol alone decreased the formation of insoluble aggregates and improved the stability of both Ant2 and Ant3. Structural models were generated for Ant2 and Ant3, and solubility-based prediction tools were employed to determine the role of hydrophobicity and charge on protein production. The results showed that a large non-polar region (containing hydrophobic amino acids) was detected on the surface of Ant2 structures, whereas positively charged regions (containing lysine and arginine amino acids) were observed for Ant3, both of which were associated with poor protein solubility. We present a guide of strategies and predictive approaches that aim to guide the construct design, prior to expression studies, to define and engineer sequences/structures that could lead to increased expression and stability of single and potentially multi-domain (or fusion) antigens in bacterial expression systems.

scholarly journals Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Liyuan Zhang ◽  
Xiaomei Lin ◽  
Ting Wang ◽  
Wei Guo ◽  
Yuan Lu
Keyword(s):  
Protein Synthesis ◽  
Protein Production ◽  
Influential Factors ◽  
Competent Cell ◽  
Free Protein ◽  
Application Range ◽  
E Coli ◽  
Short Time ◽  
Cell Free Protein Synthesis

AbstractCell-free protein synthesis (CFPS) systems have become an ideal choice for pathway prototyping, protein production, and biosensing, due to their high controllability, tolerance, stability, and ability to produce proteins in a short time. At present, the widely used CFPS systems are mainly based on Escherichia coli strain. Bacillus subtilis, Corynebacterium glutamate, and Vibrio natriegens are potential chassis cells for many biotechnological applications with their respective characteristics. Therefore, to expand the platform of the CFPS systems and options for protein production, four prokaryotes, E. coli, B. subtilis, C. glutamate, and V. natriegens were selected as host organisms to construct the CFPS systems and be compared. Moreover, the process parameters of the CFPS system were optimized, including the codon usage, plasmid synthesis competent cell selection, plasmid concentration, ribosomal binding site (RBS), and CFPS system reagent components. By optimizing and comparing the main influencing factors of different CFPS systems, the systems can be optimized directly for the most influential factors to further improve the protein yield of the systems. In addition, to demonstrate the applicability of the CFPS systems, it was proved that the four CFPS systems all had the potential to produce therapeutic proteins, and they could produce the receptor-binding domain (RBD) protein of SARS-CoV-2 with functional activity. They not only could expand the potential options for in vitro protein production, but also could increase the application range of the system by expanding the cell-free protein synthesis platform.

scholarly journals Characterization of synthetic riboswitch in cell-free protein expression systems

RNA Biology ◽  
2021 ◽  
pp. 1-12
Author(s):  
Yaroslav Chushak ◽  
Svetlana Harbaugh ◽  
Kathryn Zimlich ◽  
Bryan Alfred ◽  
Jorge Chávez ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression Systems ◽  
Free Protein

scholarly journals Unveiling Putative Functions of Mucus Proteins and Their Tryptic Peptides in Seven Gastropod Species Using Comparative Proteomics and Machine Learning-Based Bioinformatics Predictions

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3475
Author(s):  
Viroj Tachapuripunya ◽  
Sittiruk Roytrakul ◽  
Pramote Chumnanpuen ◽  
Teerasak E-kobon
Keyword(s):  
Bioactive Peptides ◽  
Nearest Neighbor ◽  
Peptide Identification ◽  
Comparative Proteomics ◽  
Mass Spectrometric ◽  
Functional Categories ◽  
K Nearest Neighbor ◽  
Gastropod Species ◽  
Bioactive Properties ◽  
Sds Page

Gastropods are among the most diverse animals. Gastropod mucus contains several glycoproteins and peptides that vary by species and habitat. Some bioactive peptides from gastropod mucus were identified only in a few species. Therefore, using biochemical, mass spectrometric, and bioinformatics approaches, this study aimed to comprehensively identify putative bioactive peptides from the mucus proteomes of seven commonly found or commercially valuable gastropods. The mucus was collected in triplicate samples, and the proteins were separated by 1D-SDS-PAGE before tryptic digestion and peptide identification by nano LC-MS/MS. The mucus peptides were subsequently compared with R scripts. A total of 2818 different peptides constituting 1634 proteins from the mucus samples were identified, and 1218 of these peptides (43%) were core peptides found in the mucus of all examined species. Clustering and correspondence analyses of 1600 variable peptides showed unique mucous peptide patterns for each species. The high-throughput k-nearest neighbor and random forest-based prediction programs were developed with more than 95% averaged accuracy and could identify 11 functional categories of putative bioactive peptides and 268 peptides (9.5%) with at least five to seven bioactive properties. Antihypertensive, drug-delivering, and antiparasitic peptides were predominant. These peptides provide an understanding of gastropod mucus, and the putative bioactive peptides are expected to be experimentally validated for further medical, pharmaceutical, and cosmetic applications.

On the dependability of highly critical non-recoverable space entities with short operation life. Case study of single-use mechanical devices

Dependability ◽  
2021 ◽  
Vol 21 (3) ◽  
pp. 3-12
Author(s):  
Yu. P. Pokhabov
Keyword(s):  
Process Engineering ◽  
Small Scale ◽  
Design Engineering ◽  
Scale Production ◽  
Decimal Point ◽  
Engineering Analysis ◽  
Single Use ◽  
Mechanical Devices ◽  
Verification Techniques

Aim. To consider matters of dependability of highly critical non-recoverable space products with short operation life, whose failures are primarily caused by design and process engineering errors, manufacturing defects in the course of single-unit or small-scale production, as well as to define the methodological approach to ensuring the required reliability.Methods. Options were analysed for improving the dependability of entities with short operation life using the case study of single-use mechanical devices and the statistical approaches of the modern dependability theory, special methods of dependability of actuated mechanical assemblies, FMEA, Stage-Gate and ground experiments on single workout equivalents for each type of effect. Results. It was concluded that additional procedures need to be conducted for the purpose of predicting, mitigation and (or) eliminating possible failures as part of the design process using exactly the same approaches that cause failures, i.e., those of design and process engineering. The engineering approaches to dependability are based on early identification of possible causes of failures, which requires a qualified and systemic analysis aimed at identifying the functionality, performance and dependability of an entity, taking into account critical output parameters and probabilistic indicators that affect the performance of the required functions with the allowable probability of failure. The solution is found using a generalized parametric model of operation and design engineering analysis of dependability.Conclusion. For highly critical non-recoverable space entities with short operation life, the reliability requirements should be considered primarily in terms financial, economic, safetyrelated and reputational risks associated with the loss of spacecraft. From a design engineer’s standpoint, the number of nines after the decimal point (rounded to a smaller number of nines for increased confidence) should be seen as the indicator for the application of the appropriate approaches to ensuring the required reliability at the stage of product design. In case of two nines after the decimal point it is quite acceptable to use analytical and experimental verification techniques common to the aerospace industry, i.e., dependability calculations using the statistical methods of the modern dependability theory and performance indicators, FMEA and Stage-Gate, ground experiments on single workout equivalents for each type of effect. As the required number of nines grows, it is advisable to also use early failure prevention methods, one of which is the design engineering analysis of dependability that enables designers to adopt substantiated design solutions on the basis of engineering disciplines and design and process engineering methods of ensuring quality and dependability. The choice of either of the above dependability strategies is determined solely by the developer’s awareness and understanding of potential hazards, which allows managing the risk of potential rare failures or reasonably refusing to do so.

scholarly journals Plant expression systems for production of hemagglutinin as a vaccine against influenza virus.

2014 ◽  
Vol 61 (3) ◽  
Author(s):  
Patrycja Redkiewicz ◽  
Agnieszka Sirko ◽  
Katarzyna Anna Kamel ◽  
Anna Góra-Sochacka
Keyword(s):  
Influenza Virus ◽  
Large Scale ◽  
Immunological Response ◽  
Biologically Active ◽  
Scale Production ◽  
Expression Systems ◽  
Lethal Challenge ◽  
Subunit Vaccines ◽  
Large Scale Production ◽  
Major Surface

Many examples of a successful application of plant-based expression systems for production of biologically active recombinant proteins exist in the literature. These systems can function as inexpensive platforms for the large scale production of recombinant pharmaceuticals or subunit vaccines. Hemagglutinin (HA) is a major surface antigen of the influenza virus, thus it is in the centre of interests of various subunit vaccine engineering programs. Large scale production of recombinant HA in traditional expression systems, such as mammalian or insect cells, besides other limitations, is expensive and time-consuming. These difficulties stimulate an ever-increasing interest in plant-based production of this recombinant protein. Over the last few years many successful cases of HA production in plants, using both transient and stable expression systems have been reported. Various forms of recombinant HA, including monomers, trimers, virus like particles (VLPs) or chimeric proteins containing its fusion with other polypeptides were obtained and shown to maintain a proper antigenicity. Immunizations of animals (mice, ferrets, rabbits or chickens) with some of these plant-derived hemagglutinin variants were performed, and their effectiveness in induction of immunological response and protection against lethal challenge with influenza virus demonstrated. Plant-produced recombinant subunit vaccines and plant-made VLPs were successfully tested in clinical trials (Phase I and II) that confirmed their tolerance and immunogenicity.

A roadmap to improve yield of the recombinant protein production: a case study employing Komagataella phaffii as the host organism

2016 ◽  
Vol 33 ◽  
pp. S49
Author(s):  
Ayca Cankorur-Cetinkaya ◽  
Duygu Dikicioglu ◽  
Joao Dias ◽  
Jana Kludas ◽  
Juho Rousu ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Host Organism ◽  
Komagataella Phaffii

Enhancing Projects Value by Embedding the Full-Scale Production System into Reservoir Model – A Case Study of Karachaganak Field, Kazakhstan

2020 ◽  
Author(s):  
Kanat Aktassov ◽  
Yerzhan Karlykhanov ◽  
Olzhas Tleukhabyluly ◽  
Kanat Imagambetov ◽  
Alexander Folefac ◽  
...  
Keyword(s):  
Production System ◽  
Full Scale ◽  
Scale Production ◽  
Reservoir Model

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..

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