scholarly journals Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains

1970 ◽  
Vol 2 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Santosh Dulal ◽  
Bhupal Ban ◽  
Gi Hyoek Yang ◽  
Hyun Ho Jung
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Clostridium Botulinum ◽  
Pathogen Detection ◽  
Recombinant Protein Expression ◽  
Synthetic Genes ◽  
Effective System ◽  
Genes Encoding

The use of recombinant BoNT domains has been proposed as a means to develop strategies to treat and prevent botulism. Here, details on the molecular cloning, protein expression, purification, and immunoreactivity of BoNT/F domains from Clostridium botulinum are presented. Initially, full-length synthetic genes encoding recombinant BoNT/F domains (catalytic, translocation, and receptor binding) were designed and cloned into Escherichia coli for expression. Recombinant proteins were then purified through GST affinity chromatography preceding elution of GST-free recombinant domains by thrombin protease. Soluble recombinant proteins encoding catalytic light chain and translocation N-terminal heavy chain were subsequently used to perform in vivo immunization. Polyclonal mouse antibodies specific to these domains were raised, confirmed by Western blot analysis and elevated immunoreactivity was identified through indirect ELISA. In conclusion, availability of the recombinant protein provides an effective system to study the immunological aspects of BoNT/F and corresponding applications in pathogen detection and vaccine candidacy. Keywords: Clostridium botulunium; Botulinum Neurotoxin Type F (BoNT/F) domains; cloning; recombinant protein expression; immunoreactivity DOI: http://dx.doi.org/10.3126/njb.v2i1.5634 Nepal Journal of Biotechnology Jan.2012, Vol.2(1): 1-15

scholarly journals Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

2020 ◽  
Vol 21 (3) ◽  
pp. 990 ◽  
Author(s):  
Kangsan Kim ◽  
Donghui Choe ◽  
Dae-Hee Lee ◽  
Byung-Kwan Cho
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Heterologous Protein ◽  
Recombinant Protein Expression ◽  
Cost Effective ◽  
Protein Yield ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins ◽  
Expression Pathways

A large proportion of the recombinant proteins manufactured today rely on microbe-based expression systems owing to their relatively simple and cost-effective production schemes. However, several issues in microbial protein expression, including formation of insoluble aggregates, low protein yield, and cell death are still highly recursive and tricky to optimize. These obstacles are usually rooted in the metabolic capacity of the expression host, limitation of cellular translational machineries, or genetic instability. To this end, several microbial strains having precisely designed genomes have been suggested as a way around the recurrent problems in recombinant protein expression. Already, a growing number of prokaryotic chassis strains have been genome-streamlined to attain superior cellular fitness, recombinant protein yield, and stability of the exogenous expression pathways. In this review, we outline challenges associated with heterologous protein expression, some examples of microbial chassis engineered for the production of recombinant proteins, and emerging tools to optimize the expression of heterologous proteins. In particular, we discuss the synthetic biology approaches to design and build and test genome-reduced microbial chassis that carry desirable characteristics for heterologous protein expression.

Recombinant Protein Expression in Escherichia coli (E.coli): What We Need to Know

2018 ◽  
Vol 24 (6) ◽  
pp. 718-725 ◽  
Author(s):  
Seyed Mohammad Gheibi Hayat ◽  
Najmeh Farahani ◽  
Behrouz Golichenari ◽  
Amirhossein Sahebkar
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Recombinant Protein Expression ◽  
Culture Conditions ◽  
Strain Vector ◽  
Factors Affecting ◽  
Expression In Escherichia Coli ◽  
Pros And Cons

Background: Host, vector, and culture conditions (including cultivation media) are considered among the three main elements contributing to a successful production of recombinant proteins. Accordingly, one of the most common hosts to produce recombinant therapeutic proteins is Escherichia coli. Methodology: A comprehensive literature review was performed to identify important factors affecting production of recombinant proteins in Escherichia coli. Results: Escherichia coli is taken into account as the easiest, quickest, and cheapest host with a fully known genome. Thus, numerous modifications have been carried out on Escherichia coli to optimize it as a good candidate for protein expression and; as a result, several engineered strains of Escherichia coli have been designed. In general; host strain, vector, and cultivation parameters are recognized as crucial ones determining success of recombinant protein expression in Escherichia coli. In this review, the role of host, vector, and culture conditions along with current pros and cons of different types of these factors leading to success of recombinant protein expression in Escherichia coli were discussed. Conclusion: Successful protein expression in Escherichia coli necessitates a broad knowledge about physicochemical properties of recombinant proteins, selection among common strains of Escherichia coli and vectors, as well as factors related to media including time, temperature, and inducer.

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 Multimodal Approaches for the Improvement of the Cellular Folding of a Recombinant Iron Regulatory Protein in E. Coli

2021 ◽  
Author(s):  
Gayathri Ravitchandirane ◽  
Sheetal Bandhu ◽  
Tapan K. Chaudhuri
Keyword(s):  
Heat Shock ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Physiological Stress ◽  
Recombinant Protein Expression ◽  
Host Cells ◽  
Growth Media ◽  
E Coli ◽  
Expression Host

Abstract BackgroundDuring the recombinant protein expression, foreign proteins are generated in insoluble and inactive aggregates in E. coli cell factories, which inhibits E. coli from being employed as an expression host despite its numerous advantages and ease of use. The yeast mitochondrial aconitase protein, which has a tendency to aggregate when expressed in E. coli cells in the absence of heterologous chaperones GroEL/ES was utilised as a model to investigate how the modulation of physiological stimuli in the host cell can increase protein solubility. The process variables such as incubation temperature, inducer concentrations, growth media, and the presence of folding modulators such as exogenous molecular chaperones or osmolytes are crucial for the cellular folding and are investigated in the study. The processes the physiological stress such as osmotic and heat shock stimulation in the host cells and thereby their effect on the solubility and activity of recombinant proteins was also analysed.ResultsOf the various methods discussed, the cells subjected to the addition of osmolytes and pre-induction heat shock exhibited significant enhancement in the recombinant aconitase activity. The concomitant GroEL/ES expression further assists the folding of these soluble aggregates and increases the functional protein molecules in the cytoplasm of the recombinant E. coli cells.ConclusionsThe recombinant E. coli cells enduring physiological stress provide a cytosolic environment for the enhancement in the solubility and activity of the recombinant proteins. GroEL/ES-expressing cells not only aided in the folding of recombinant proteins, but also had an effect on the physiology of the expression host. The improvement in the specific growth rate and aconitase productivity during chaperone GroEL/ES co-expression is attributed to the reduction in overall cellular stress caused by the expression host's aggregation-prone recombinant protein expression.

scholarly journals High-Throughput Screening Identifies Two Novel Small Molecule Enhancers of Recombinant Protein Expression

Molecules ◽  
2020 ◽  
Vol 25 (2) ◽  
pp. 353
Author(s):  
Jiasong Chang ◽  
Xiaoxu Chen ◽  
Ruolin Wang ◽  
Run Shi ◽  
Xiaogang Wang ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Small Molecules ◽  
Small Molecule ◽  
Recombinant Proteins ◽  
High Throughput ◽  
High Throughput Screening ◽  
Histone Deacetylase Inhibitors ◽  
Recombinant Protein Expression ◽  
Expression Levels

As a primary strategy for production of biological drugs, recombinant proteins produced by transient transfection of mammalian cells are essential for both basic research and industrial production. Here, we established a high-throughput screening platform for improving the expression levels of recombinant proteins. In total, 10,011 small molecule compounds were screened through our platform. After two rounds of screening, we identified two compounds, Apicidin and M-344, that significantly enhanced recombinant protein expression. Both of the selected compounds were histone deacetylase inhibitors, suggesting that the two small molecules increased the expression levels of recombinant proteins by promoting histone acetylation. Moreover, both molecules showed low cytotoxicity. Therefore, our findings suggest that these small molecules may have wide applications in the future.

scholarly journals Choice of selectable marker affects recombinant protein expression in cells and exosomes

2021 ◽  
pp. 100838
Author(s):  
Chenxu Guo ◽  
Francis K. Fordjour ◽  
Shang Jui Tsai ◽  
James C. Morrell ◽  
Stephen J. Gould
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Selectable Marker ◽  
Recombinant Protein Expression ◽  
In Cells

BacMam System for Rapid Recombinant Protein Expression in Mammalian Cells

2019 ◽  
pp. 205-208 ◽  
Author(s):  
Deepak B. Thimiri Govinda Raj ◽  
Niamat Ali Khan ◽  
Srisaran Venkatachalam ◽  
Sivakumar Arumugam
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Mammalian Cells ◽  
Recombinant Protein Expression ◽  
Expression In Mammalian Cells

Evaluation of somatic embryos of alfalfa for recombinant protein expression

2014 ◽  
Vol 34 (2) ◽  
pp. 211-221 ◽  
Author(s):  
Guohua Fu ◽  
Vojislava Grbic ◽  
Shengwu Ma ◽  
Lining Tian
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Somatic Embryos ◽  
Recombinant Protein Expression

scholarly journals Mutation of Putative N-Glycosylation Sites on Dengue Virus NS4B Decreases RNA Replication

2015 ◽  
Vol 89 (13) ◽  
pp. 6746-6760 ◽  
Author(s):  
Nenavath Gopal Naik ◽  
Huey-Nan Wu
Keyword(s):  
Life Cycle ◽  
Recombinant Protein ◽  
Dengue Virus ◽  
Protein Expression ◽  
Nonstructural Protein ◽  
Recombinant Protein Expression ◽  
Rna Replication ◽  
Viral Production ◽  
Glycosylation Sites ◽  
Infected Cells

ABSTRACTDengue virus (DENV) nonstructural protein 4B (NS4B) is an endoplasmic reticulum (ER) membrane-associated protein, and mutagenesis studies have revealed its significance in viral genome replication. In this work, we demonstrated that NS4B is an N-glycosylated protein in virus-infected cells as well as in recombinant protein expression. NS4B is N glycosylated at residues 58 and 62 and exists in two forms, glycosylated and unglycosylated. We manipulated full-length infectious RNA clones and subgenomic replicons to generate N58Q, N62Q, and N58QN62Q mutants. Each of the single mutants had distinct effects, but the N58QN62Q mutation resulted in dramatic reduction of viral production efficiency without affecting secretion or infectivity of the virion in mammalian and mosquito C6/36 hosts. Real-time quantitative PCR (qPCR), subgenomic replicon, andtrans-complementation assays indicated that the N58QN62Q mutation affected RNA replication possibly by the loss of glycans. In addition, four intragenic mutations (S59Y, S59F, T66A, and A137T) were obtained from mammalian and/or mosquito C6/36 cell culture systems. All of these second-site mutations compensated for the replication defect of the N58QN62Q mutant without creating novel glycosylation sites.In vivoprotein stability analyses revealed that the N58QN62Q mutation alone or plus a compensatory mutation did not affect the stability of NS4B. Overall, our findings indicated that mutation of putative N-glycosylation sites affected the biological function of NS4B in the viral replication complex.IMPORTANCEThis is the first report to identify and reveal the biological significance of dengue virus (DENV) nonstructural protein 4B (NS4B) posttranslation N-glycosylation to the virus life cycle. The study demonstrated that NS4B is N glycosylated in virus-infected cells and in recombinant protein expression. NS4B is modified by glycans at Asn-58 and Asn-62. Functional characterization implied that DENV NS4B utilizes the glycosylation machinery in both mammalian and mosquito hosts. Four intragenic mutations were found to compensate for replication and subsequent viral production deficiencies without creating novel N-glycosylation sites or modulating the stabilities of the protein, suggesting that glycans may be involved in maintaining the NS4B protein conformation. NS4B glycans may be necessary elements of the viral life cycle, but compensatory mutations can circumvent their requirement. This novel finding may have broader implications in flaviviral biology as the most likely glycan at Asn-62 of NS4B is conserved in DENV serotypes and in some related flaviviruses.

Sign in / Sign up

Export Citation Format

Share Document