scholarly journals A positive Selection Escherichia Coli Recombinant Protein Expression Vector for One-Step Cloning

2022 ◽  
Vol 9 ◽  
Author(s):  
Shinto James ◽  
Vikas Jain
Keyword(s):  
Recombinant Protein ◽  
Fluorescent Protein ◽  
Recombinant Protein Expression ◽  
Plasmid Vector ◽  
Cost Effective ◽  
Single Step ◽  
Selection Strategy ◽  
Host Bacterium ◽  
T7 Promoter ◽  
Vector Systems

We introduce OLIVAR (Orientation seLection of Insert in Vector through Antisense Reporter) as a novel selection strategy for the insertion of protein-coding genes into vector backbones. As a proof-of-concept, we have engineered a plasmid vector, pGRASS (Green fluorescent protein Reporter from Antisense promoter-based Screening System), for gene cloning in E. coli. With pGRASS, positive clones can be effortlessly distinguished from negative clones after blunt-end cloning. The vector not only screens clones with an insert but also for its correct orientation. The design further allows for the expression of recombinant protein from the T7 promoter in an appropriate host bacterium. With this vector, we are able to reduce the entire cloning workflow into a single step involving a 2-h reaction at room temperature. We believe that our cloning-cum-screening system presented here is extremely cost-effective and straightforward and can be applied to other vector systems and domains such as phage display and library construction.

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.

scholarly journals Effects of Recombinant Protein Expression on Green Fluorescent Protein Diffusion inEscherichia coli

Biochemistry ◽  
2009 ◽  
Vol 48 (23) ◽  
pp. 5083-5089 ◽  
Author(s):  
Kristin M. Slade ◽  
Rachael Baker ◽  
Michael Chua ◽  
Nancy L. Thompson ◽  
Gary J. Pielak
Keyword(s):  
Green Fluorescent Protein ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Fluorescent Protein ◽  
Recombinant Protein Expression ◽  
Inescherichia Coli ◽  
Protein Diffusion ◽  
Green Fluorescent

scholarly journals Integrated Recombinant Protein Expression and Purification Platform Based on Ralstonia eutropha

2005 ◽  
Vol 71 (10) ◽  
pp. 5735-5742 ◽  
Author(s):  
Gavin C. Barnard ◽  
Jesse D. McCool ◽  
David W. Wood ◽  
Tillman U. Gerngross
Keyword(s):  
Protein Purification ◽  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Recombinant Protein Expression ◽  
Specific Interaction ◽  
Single Step ◽  
Protein Expression And Purification ◽  
Model Protein ◽  
Purification Methods ◽  
Green Fluorescent

ABSTRACT Protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. We describe a protein purification scheme wherein Ralstonia eutropha is used to produce its own “affinity matrix,” thereby eliminating the need for external chromatographic purification steps. This approach is based on the specific interaction of phasin proteins with granules of the intracellular polymer polyhydroxybutyrate (PHB). By creating in-frame fusions of phasins and green fluorescent protein (GFP) as a model protein, we demonstrated that GFP can be efficiently sequestered to the surface of PHB granules. In a second step, we generated a phasin-intein-GFP fusion, wherein the self-cleaving intein can be activated by the addition of thiols. This construct allowed for the controlled binding and release of essentially pure GFP in a single separation step. Finally, pure, active β-galactosidase was obtained in a single step using the above described method.

A Comprehensive Guide to the Commercial Baculovirus Expression Vector Systems for Recombinant Protein Production

2020 ◽  
Vol 27 (6) ◽  
pp. 529-537 ◽  
Author(s):  
Vibhor Mishra
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Expression Vector ◽  
Recombinant Protein Expression ◽  
Scale Up ◽  
Vector System ◽  
Heterologous Protein Expression ◽  
Baculovirus Expression ◽  
Vector Systems ◽  
Baculovirus Expression Vector

The Baculovirus Expression Vector System (BEVS) is a workhorse for recombinant protein expression for over thirty-five years. Ever since it was first used to overexpress the human IFN-β protein, the system has been engineered and modified several times for quick and easy expression and scale-up of the recombinant proteins. Multiple gene assemblies performed on the baculovirus genome using synthetic biology methods lead to optimized overexpression of the multiprotein complexes. Nowadays, several commercially available BEVS platforms offer a variety of customizable features, and often it is confusing which one to choose for a novice user. This short review is intended to be a one-stop guide to the commercially available baculovirus technology for heterologous protein expression in the insect cells, which users can refer to choose from popular and desirable BEVS products or services.

scholarly journals Overview of Bacterial and Yeast Systems for Protein Expression

2021 ◽  
pp. 113-118
Author(s):  
Maheswara Reddy Mallu ◽  
Siva Reddy Golamari ◽  
Sree Rama Chandra Karthik Kotikalapudi ◽  
Renuka Vemparala
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Large Scale ◽  
Recombinant Protein Expression ◽  
Expression Vectors ◽  
Scale Production ◽  
Protein Coding ◽  
Recombinant Gene Expression ◽  
Vector Systems ◽  
Large Scale Production

Over the past decade the variety of hosts and vector systems for recombinant protein expression has increased dramatically. Researchers now select from among mammalian, insect, yeast, and prokaryotic hosts, and the number of vectors available for use in these organisms continues to grow. With the increased availability of cDNAs and protein coding sequencing information, it is certain that these and other, yet to be developed systems will be important in the future. Despite the development of eukaryotic systems, E. coli remains the most widely used host for recombinant protein expression. Optimization of recombinant protein expression in prokaryotic and eukaryotic host systems has been carried out by varying simple parameters such as expression vectors, host strains, media composition, and growth temperature. Recombinant gene expression in eukaryotic systems is often the only viable route to the large-scale production of authentic, post translationally modified proteins. It is becoming increasingly easy to find a suitable system to overexpress virtually any gene product, provided that it is properly engineered into an appropriate expression vector.

scholarly journals Recombinant Protein Production with Escherichia coli in Glucose and Glycerol Limited Chemostats

2021 ◽  
Vol 1 (2) ◽  
pp. 239-254
Author(s):  
Anca Manuela Mitchell ◽  
Valentina Gogulancea ◽  
Wendy Smith ◽  
Anil Wipat ◽  
Irina Dana Ofiţeru
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Fluorescent Protein ◽  
Recombinant Protein Expression ◽  
Coli Strain ◽  
Biodiesel Production ◽  
Biological Drugs

Recently, there has been a resurgence of interest in continuous bioprocessing as a cost-optimised production strategy, driven by a rising global requirement for recombinant proteins used as biological drugs. This strategy could provide several benefits over traditional batch processing, including smaller bioreactors, smaller facilities, and overall reduced plant footprints and investment costs. Continuous processes may also offer improved product quality and minimise heterogeneity, both in the culture and in the product. In this paper, a model protein, green fluorescent protein (GFP) mut3*, was used to test the recombinant protein expression in an Escherichia coli strain with industrial relevance grown in chemostat. An important factor in enabling stable productivity in continuous cultures is the carbon source. We have studied the viability and heterogeneity of the chemostat cultures using a chemically defined medium based on glucose or glycerol as the single carbon source. As a by-product of biodiesel production, glycerol is expected to become a sustainable alternative substrate to glucose. We have found that although glycerol gives a higher cell density, it also generates higher heterogeneity in the culture and a less stable recombinant protein production. We suggest that manipulating the balance between different subpopulations to increase the proportion of productive cells may be a possible solution for making glycerol a successful alternative to glucose.

scholarly journals Novel Regeneration Approach for Creating Reusable FO-SPR Probes with NTA Surface Chemistry

Nanomaterials ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 186
Author(s):  
Jia-Huan Qu ◽  
Karen Leirs ◽  
Remei Escudero ◽  
Žiga Strmšek ◽  
Roman Jerala ◽  
...  
Keyword(s):  
Surface Chemistry ◽  
Fluorescent Protein ◽  
Cost Effective ◽  
Nitrilotriacetic Acid ◽  
Antibody Fragments ◽  
Rapid Screening ◽  
Red Fluorescent Protein ◽  
Effective Manner ◽  
Surface Regeneration ◽  
Sensitivity Specificity

To date, surface plasmon resonance (SPR) biosensors have been exploited in numerous different contexts while continuously pushing boundaries in terms of improved sensitivity, specificity, portability and reusability. The latter has attracted attention as a viable alternative to disposable biosensors, also offering prospects for rapid screening of biomolecules or biomolecular interactions. In this context here, we developed an approach to successfully regenerate a fiber-optic (FO)-SPR surface when utilizing cobalt (II)-nitrilotriacetic acid (NTA) surface chemistry. To achieve this, we tested multiple regeneration conditions that can disrupt the NTA chelate on a surface fully saturated with His6-tagged antibody fragments (scFv-33H1F7) over ten regeneration cycles. The best surface regeneration was obtained when combining 100 mM EDTA, 500 mM imidazole and 0.5% SDS at pH 8.0 for 1 min with shaking at 150 rpm followed by washing with 0.5 M NaOH for 3 min. The true versatility of the established approach was proven by regenerating the NTA surface for ten cycles with three other model system bioreceptors, different in their size and structure: His6-tagged SARS-CoV-2 spike fragment (receptor binding domain, RBD), a red fluorescent protein (RFP) and protein origami carrying 4 RFPs (Tet12SN-RRRR). Enabling the removal of His6-tagged bioreceptors from NTA surfaces in a fast and cost-effective manner can have broad applications, spanning from the development of biosensors and various biopharmaceutical analyses to the synthesis of novel biomaterials.

scholarly journals 38 Using pooled data for single-step genomic prediction: Impact of within-pool variance and size

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 9-9
Author(s):  
Johnna L Baller ◽  
Stephen D Kachman ◽  
Larry A Kuehn ◽  
Matthew L Spangler
Keyword(s):  
Phenotypic Variation ◽  
Equal Opportunity ◽  
Cost Effective ◽  
Single Step ◽  
Pool Size ◽  
Individual Data ◽  
Phenotypic Data ◽  
Breeding Values ◽  
Mean Values ◽  
Genetic Evaluations

Abstract Economically relevant traits (ERT) are routinely collected within commercial segments of the beef industry but are rarely included in genetic evaluations because of unknown pedigrees. Individual relationships could be resurrected with genomics, which would be costly; pooling DNA and phenotypic data provides a cost-effective solution. A simulated beef cattle population consisting of 15 generations was genotyped with approximately 50k markers (841 quantitative trait loci were located across the genome) and phenotyped for a moderately heritable trait. Individuals from generation 15 were included in pools (observed genotype and phenotype were mean values of a group). Estimated breeding values (EBV) were generated from a single-step GBLUP model. The effects of pooling strategy (random and minimizing or uniformly maximizing phenotypic variation), pool size (1, 2, 10, 20, 50, 100, or no data from generation 15), and generational gaps of genotyping on EBV accuracy (correlation of EBV with true breeding values) were quantified. Greatest EBV accuracies of sires and dams were observed when no gap between genotyped parents and pooled offspring occurred. The EBV accuracies resulting from pools were greater than no data from generation 15 regardless of sire or dam genotyping. Minimizing phenotypic variation increased EBV accuracy by 8% and 9% over random pooling and uniformly maximizing phenotypic variation, respectively. Pool size of 2 was the only scenario that did not significantly decrease EBV accuracy compared to individual data when pools were formed randomly or by uniformly maximizing phenotypic variation (P > 0.05). Pool sizes of 2, 10, 20, or 50 did not generally lead to EBV accuracies that were statistically different than individual data when pools were constructed to minimize phenotypic variation (P > 0.05). Pooled genotyping to garner commercial-level phenotypes for genetic evaluations seems plausible, although differences exist depending on pool size and pool formation strategy. The USDA is an equal opportunity employer.

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