The generation of a stable master cell lineEchoHan4
The generation of a stable master cell line requires the integration of the expression cassette into the host cell genome.
https://www.creativebiolabs.net/magic-mammalian-cell-expression-service.htm
Expression and purification of recombinant proteins in Bacterial and yeast sy...Shreya Feliz
This presentation gives the information about bacterial and yeast system as host for expressing recombinant proteins, suitable vectors, strains of host, Pros and cons of this system, different purification techniques and commercially available proteins produced so far by this system.
The generation of a stable master cell lineEchoHan4
The generation of a stable master cell line requires the integration of the expression cassette into the host cell genome.
https://www.creativebiolabs.net/magic-mammalian-cell-expression-service.htm
Expression and purification of recombinant proteins in Bacterial and yeast sy...Shreya Feliz
This presentation gives the information about bacterial and yeast system as host for expressing recombinant proteins, suitable vectors, strains of host, Pros and cons of this system, different purification techniques and commercially available proteins produced so far by this system.
Cell culture based vaccine??
Cell cultures involve growing cells in a culture dish, often with a supportive growth medium. A primary cell culture consists of cells taken directly from living tissue, and may contain multiple types of cells such as fibroblasts, epithelial, and endothelial cells.
In the United States, 10 different vaccines for chicken pox, hepatitis A, polio, rabies, and rubella are cultured on aborted tissue from two fetal cell lines known as WI-38 and MRC-5. These vaccines are chicken pox, hep-A, hep-A, hep-A/hep-B, polio, rabies, rubella, measles/rubella, mumps/rubella, and MMR II (measles/mumps/rubella).
Need to revolutionize the crop breedingamoldchokhat
Mankind is facing an enormous challenge of food insecurity. By the year 2050, the population of the world is projected to increase by 35% to a whopping 9 billion; and an almost doubling of global food production is needed to feed the planet. This additional food for humans and farm animals has to come from the existing land, through maximization of genetic potential and relatively quickly. This demand for unprecedented productivity in agriculture needs to be realized in the presence of growing challenges of climate change, shortsighted land-use practices and increasing cost of agriculture despite the rate of improved adoption of technology in crop breeding. Recent advances in our understanding of genes and genomes combined with development of novel tools in biotechnology will play a vital role in accelerating efforts in plant breeding. Genomics assisted breeding assists the breeders in precise selection to enhance the effectivity and enhancement of the precise selection to develop a new cultivars.
Cow´s milk allergy is a common food allergy especially among infants and young children. The major allergen in cow´s milk is beta-lactoglobulin (BLG) and BLG-specific antibodies are needed for diagnostic, therapeutic, food processing and quality verification applications. At VTT we have established a IgE antibody library from a milk allergic person and identified high-affinity antibodies against native and heat denaturated BLG. As an alternative production system, we have used barley as a production host. Proof-of-concept was gained with expressing the BLG-specific antibody in barley cell culture. Best production levels in barley grains were obtained with glutelin-specific promoter combined to ER targeting and retention signals. The barley-produced BLG-specific antibody was successfully purifed with affinity-based chromatography and the functionality of barley-produced BLG-specific antibody was verified by ELISA and immunoprecipitation assays. The developed barley-based expression system clearly demonstrated its potential for application in the processing of dairy milk products as well as in detecting allergens from foods possibly contaminated by cow´s milk.
Cell culture based vaccine??
Cell cultures involve growing cells in a culture dish, often with a supportive growth medium. A primary cell culture consists of cells taken directly from living tissue, and may contain multiple types of cells such as fibroblasts, epithelial, and endothelial cells.
In the United States, 10 different vaccines for chicken pox, hepatitis A, polio, rabies, and rubella are cultured on aborted tissue from two fetal cell lines known as WI-38 and MRC-5. These vaccines are chicken pox, hep-A, hep-A, hep-A/hep-B, polio, rabies, rubella, measles/rubella, mumps/rubella, and MMR II (measles/mumps/rubella).
Need to revolutionize the crop breedingamoldchokhat
Mankind is facing an enormous challenge of food insecurity. By the year 2050, the population of the world is projected to increase by 35% to a whopping 9 billion; and an almost doubling of global food production is needed to feed the planet. This additional food for humans and farm animals has to come from the existing land, through maximization of genetic potential and relatively quickly. This demand for unprecedented productivity in agriculture needs to be realized in the presence of growing challenges of climate change, shortsighted land-use practices and increasing cost of agriculture despite the rate of improved adoption of technology in crop breeding. Recent advances in our understanding of genes and genomes combined with development of novel tools in biotechnology will play a vital role in accelerating efforts in plant breeding. Genomics assisted breeding assists the breeders in precise selection to enhance the effectivity and enhancement of the precise selection to develop a new cultivars.
Cow´s milk allergy is a common food allergy especially among infants and young children. The major allergen in cow´s milk is beta-lactoglobulin (BLG) and BLG-specific antibodies are needed for diagnostic, therapeutic, food processing and quality verification applications. At VTT we have established a IgE antibody library from a milk allergic person and identified high-affinity antibodies against native and heat denaturated BLG. As an alternative production system, we have used barley as a production host. Proof-of-concept was gained with expressing the BLG-specific antibody in barley cell culture. Best production levels in barley grains were obtained with glutelin-specific promoter combined to ER targeting and retention signals. The barley-produced BLG-specific antibody was successfully purifed with affinity-based chromatography and the functionality of barley-produced BLG-specific antibody was verified by ELISA and immunoprecipitation assays. The developed barley-based expression system clearly demonstrated its potential for application in the processing of dairy milk products as well as in detecting allergens from foods possibly contaminated by cow´s milk.
Protein expression and purification services from creative biomartAnne Ehlert
Creative BioMart is committed to providing advanced tools for protein expression and purification. As a leading supplier for reagents in the biotechnology field, we understand the importance of convenient and easy-to-use systems for high level expression and sample purification. We invite you to review our growing range of expression systems resulting from our experience in cloning, overexpression and purification.
Recombinant yeast technology at the cutting edge robust tools for both design...NavPrabh Sandhu Johal
Recombinant DNA technology is major DNA-based tool that has gained popular attention in the past decade. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of yeast species have led the recombinant yeast technology a robust tool for both designed catalysts and new biologicals. Yeast combines molecular genetic manipulations and growth characteristics of prokaryotic organisms together with the sub-cellular machinery for performing post-translational protein modifications (O and N- linked glycosylation, disulphide bond formation) and secretion of protein (Intracellularly or extracellularly). A large number of yeast hosts (Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Yarrowia lipolytica, etc) are available for heterologous protein expression. The methylotrophic yeast, Pichia species is the most highly developed one among a small group of alternative yeast species chosen for their perceived advantages over S. cerevisiae as a expression host for the generation of recombinant protein of commercial interest. Advantages of the system include the AOX1 promoter (alcohol oxidase) and other alternate promoters (GAP, FLD1, PEX8, and YPT1), with transcription characteristics that are useful for regulating heterologous protein expression.
Auxotrophic mutants (MutS and Mut+) and a new set of biosynthetic markers such as ADE1, ARG4 and URA 3 have been used successfully for better selection of transformed host. Protease deficient hosts and site specific integration of expression vectors into Pichia genome result into high expression of gene of interest. Additional features that are present in certain P. pastoris expression vectors serve as tools for specialized functions. The availability of the expression system as a commercially available kit (Invitrogen) extends the usefulness of system. Several different secretion signal sequences including the native secretion signal or secretion signal sequences from S. cerevisiae such as µ factor prepro peptide causes the protein to be secreted into the growth medium, which greatly facilitates subsequent protein purification. The P. pastoris expression platform is now well developed, as proven by multiple products used in human and veterinary medicine and in industry. A better understanding of secretion signals, glycosylation, and endogenous yeast proteases would be extremely helpful in developing and improving the yeast heterologous expression system.
Producing proteins or other metabolites useful to business or medicine in plants that are typically used in agriculture is known as molecular farming.
The practise of using plants to create recombinant protein products is known as molecular farming. The technology is now older than 30 years. The initial promise of molecular farming was predicated on three anticipated benefits: the low cost of plant cultivation, the enormous scalability of agricultural output, and the intrinsic safety of plants as hosts for the synthesis of medicines. As a result, a tonne of studies were published in which various proteins were expressed in various plant-based systems, and several businesses were established in an effort to commercialise the novel technology. For businesses making proteins for non-pharmaceutical uses, there was a modicum of success, but in the pharmaceutical industry, the hopes sparked by early, promising research were quickly dashed by the hard facts of industrial pragmatism.
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Host systems for protein expression
1. Model Host Systems
Choice of host depends on several needs:
Costs and availability of supports (kits, vectors, reagents and expertise)
Yield – large mass of protein presents specific challenges for scale up (bacteria/yeast vs.
mammalian)
How will the protein be used after expression?
Toxicity of protein on host cell
Post-translational modification
Impact on host cell on protein solubility and hydrolysis
Location of protein within the host cell – membrane, inclusion body, secretion into media
Complexity of medium
(A) E. coli
Advantages:
First choice due to level of support (technical, literature, kits, plasmids, reagents)
High yield and easier scale up (up to 100 mg per liter of culture)
Cost of media and reagents is relatively inexpensive
Many expression plasmids – many with gene expression regulation
Fast growth condition – one to two days from starter culture to cell pellet
Up to 50% of protein can be target protein
Simple culture conditions
Simple transformation protocols
Many parameters can be altered to optimize expression
Disadvantages:
Minimal post-translational modification
Lipid modification, phosphorylation, glycosylation, decarboxylation, acetylation
can not be performed, these may be critical requirements for functional protein
Large proteins may be difficult to express (plasmid size is limited to about 15 kb)
Poor folding of proteins in the cytoplasm
In vitro refolding protocols are inefficient in this system
2. Large number of proteins are toxic to E.coli
Inclusion body packing of protein
Insoluble, aggregated and non-native protein
Often times takes place in high expressed or toxic proteins
Codon Use differences, false stops and low expression
Inefficient disulfide bond formation
Endotoxin production
Example of expression vector used: pET series, pGEX series, pBAD
(B) Fungi
(I) Saccharomyces cerevisae
Advantages:
Good expression levels
Choice of secreted or cellular expression
Low cost
Simple culture conditions
Scalable
Able to perform most eukaryotic post-translational modifications
Efficient protein folding
Endotoxin free
Disadvantages:
Likely lower expression than Pichia pastoris
Secretion likely lower than Pichia pastoris
Glycosylation still different to mammalian cells
A tendency to hyperglycosylate proteins
N-glycan structures considered allergenic
3. (II) Pichia pastoris
Advantages:
High expression levels
Low cost
Simple culture conditions
Relatively rapid growth
Scalable
Choice of secreted or intracellular expression
Protein secretion efficient and allows simple purification
Extensive post-translational modification of proteins
Efficient protein folding
N-glycosylation more like higher eukaryotes than with Saccharomyces cerevisiae
Endotoxin free
Disadvantages:
Use of methanol as inducer is a safety (fire) hazard at scale
Glycosylation still different to mammalian cells
Example of expression vector used: pAO815, pGAPZ, pPIC series, pHIL series
(C) Mammalian cell lines
Advantages:
Good expression levels
Moderately scalable
Suspension-adapted cells facilitate scale-up
Efficient protein folding
Good for secreted proteins
All post-translational modifications
Endotoxin free
Disadvantages:
Expensive culture media
Complex growth requirements
4. Example of expression vector used: pcDNA series, pCMV series, pJRed series
(D) Insect cells (Baculovirus infected insect cells)
Advantages:
Good expression levels (especially for intracellular proteins)
Relatively rapid growth
Efficient protein folding
Moderately scalable
Extensive post-translational modification of proteins
Glycosylation more like mammalian cells
Relatively deglycosylate protein enzymatically (good for structure determination)
Endotoxin free
Disadvantages:
Expensive culture media
Large scale of virus needed on scale-up
Inefficient processing of pro-peptides in the secretory pathway
Glycosylation still different to mammalian cells
Viral infection leads to cell lysis and potential degradation of expressed proteins
Example of expression vector used: pAcP series, pBAC series, pAcUW31
(E) Transgenic plants:
Advantages:
Plants contain no known human pathogens (such as prions, virions, etc.) that could
contaminate the final product
Higher plants generally synthesize proteins from eukaryotes with correct folding,
glycosylation & activity
Significantly lower production cost than with transgenic animals, fermentation or
bioreactors
Reducing the costs of storage and transportation of recombinant proteins (when they are
produced in dry textures like grains)
5. Low ethical concerns
Easier purification (homologs don’t pose any purification challenge, e.g. serum proteins
or antibodies)
Versatile (production of a broad diversity of proteins)
Disadvantages:
Take more time to develop
Transgene & protein pollution
(F) Transgenic animals:
Advantages:
Low cost of production
More efficient system
Higher stability of proteins
All the post-translational modifications
Reduced risk of immunogenicity
No or reduced aseptic conditions required
Disadvantages:
Limited by legal and ethical restrictions
Health related complications in transgenic animals
Low rate of success
Prior extensive knowledge required
Potential pathogenic infection
Relatively time consuming
Scaling up problems