1. What are CUSABIO's Five Expression Systems?
Over more than 18 years, CUSABIO Protein Expression Platform has established five recombinant expression systems, which includes Escherichia coli (E.coli) expression system, Pichia pastoris (Yeast) expression system, Baculovirus-infected insect cells expression system, Mammalian cells expression system and vitro E.coli expression system.
2. Vitro E.coli Expression system, which is CUSABIO's Unique Expression System
Vitro E.coli expression system, also known as the vitro translation system, simulates the life phenomena of biological cells and reproduces the transcription and translation process of intracellular proteins. Common components of a cell-free production include a cell extract, an energy source, a supply of amino acids, cofactors such as magnesium, and the DNA/mRNA with the desired genes which is the template of protein synthesis. The vitro protein synthesis of the target protein can be achieved by artificially controlling the substrates required for protein synthesis and transcription and translation-related protein factors.
CUSABIO's cell-free expression platform can provide you with complete technical services. It can solve specific problems related to protein expression, such as low protein yield, expression of special proteins (such as membrane proteins, toxic proteins, etc.), protein complexes production, parallel synthesis of many different proteins.
There are some features of vitro E.coli expression system as follows:
A. Compared with the traditional protein expression system, many processes are omitted, such as transformation of plasmids, cell culture, collection, crushing and centrifugation etc., which greatly improves the working efficiency;
B. The reaction system is small and can simultaneously parallel synthesizing many different proteins;
C. Short reaction cycles, meeting the scientific requirements for high-throughput ligand screening and proteomics;
D. The open reaction system is more easy change the reaction conditions and is conducive to the regulation of gene transcription, protein synthesis and post-translational modifications, and avoids the formation of inclusion bodies;
E. Stable reaction system, can be coupled with other processes to form automated, procedural, and scale production and accelerate the purification, functional characterization and subsequent structural analysis of recombinant proteins;
F. No cell structures restriction, can be used to produce exogenous proteins that are toxic to the host as it seeks to avoid the lethal effect of protein expression on host cells;
G. Add unnatural amino acids or isotopically labeled amino acids to express specific proteins.
H. Significant improvements have been made in items that have difficulty to express in common cell lines due to multiple transmembrane or hydrophobic conditions.
3. The Characteristics of Five Expression Systems
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Escherichia coli (E.coli) expression system
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Advantages
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Clear genetic background;
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Low cost and simple culture conditions;
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Simple transformation operation;
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Short protein expression period;
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High rate of reproduction and expression level;
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A variety of carriers and fusion tags to choose;
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Many parameters can be altered to optimize expression.
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Tag can be available at both N-terminal and C-terminal with an enzyme cutting site that is easy to cut.
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Disadvantages
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Inefficient disulfide bonds formation and poor folding lead to inclusion body formation;
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Poor post-translational modifications (such as glycosylation, alkylation, phosphorylation, specificity of proteolytic processing;
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The success rate and recovery rate of inclusion body renaturation is not very high.
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Applications
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Purified protein (structure, enzymology, drug discovery);
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Protein therapeutics.
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Pichia pastoris (Yeast) expression system
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Advantages
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Low cost and high expression level;
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Self-produced Endotoxin-free;
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Efficient protein folding and secretory expression;
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Simple culture conditions and purification operation;
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Extensive post-translational modifications: Glycosylation, phosphorylation, acyl lipid;
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Can develop high density fermentation by using simple inorganic salt, large biomass;
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The protein is more stable than the prokaryotic expression, and is especially suitable for the expression of eukaryotic genes and the preparation of functional expression proteins;
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Simple laboratory and industrial operation.
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Disadvantages
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Glycosylation modification is not good as mammalian cells
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Applications
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Purified protein (structure, enzymology, drug discovery).
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Baculovirus-infected insect cells expression system
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Advantages
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Good expression levels and relatively rapid growth;
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More advantages over the expression of viral proteins;
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Glycosylation modification more like mammalian cells;
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Relatively de-glycosylation modification (good for structure determination);
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Large gene volume: because the baculovirus genome is larger, so it can carry large gene fragment;
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High security: the baculovirus has a strict species specificity, not infected with vertebrates and plants;
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High expression efficiency of exogenous gene: compared with other eukaryotic expression systems, the baculovirus system can efficiently express exogenous protein from infected cells;
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The expression product has high activity: baculovirus will proliferation in insect host cells, thus resulting recombinant protein will be similar to mammalian cells in post-translational modification, the expression product has a strong biological activity;
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Easy to amplify: baculovirus can mass production of recombinant protein products with biological activity.
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Disadvantages
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Expensive culture media cost;
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Inefficient processing of pro-peptides in secretory pathway;
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Viral infection leads to cell lysis and potential degradation of expressed proteins;
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Glycosylation modification is not good as mammalian cells.
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Applications
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Purified protein (structure, enzymology, drug discovery).
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Mammalian cells expression system
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Advantages
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Self-produced Endotoxin-free;
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Efficient protein folding and secretory expression;
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All post-translational modifications, provide a variety of complex N glycosylation and accurate O type glycosylation and other post-translational processing functions;
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The expressed products are closest to the natural biological proteins in molecular structure, physical and chemical properties and biological functions.
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Disadvantages
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Expensive culture media cost;
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Complex growth requirements.
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Applications
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Purified protein (structure, enzymology, drug discovery);
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Protein therapeutics;
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Cell-based studies.
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vitro E.coil expression system
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Advantages
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High expression level: we provide the original and optimized pET carrier with a series of labels, so that the membrane protein yield can reach mg grades;
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Protein synthesis conditions can be manipulated;
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The target fusion protein with 6*His is beneficial to the purification.
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Disadvantages
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Limited post-translational modifications;
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Expensive for scale-up.
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Applications
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Purified protein (structure, enzymology, drug discovery);
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In vitro expression cloning;
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Isotopic labelling of proteins for NMR;
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Incorporation of non-natural amino acids.
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4. How to Choose The Expression System according to The Type of Protein?
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Expression System
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System Benefits
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Application
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Features of CUSABIO
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In vitro E.coli Expression System
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Simple, take short time, high expression quantity, open and flexible, easy to express specific proteins, prepare protein complexes, parallel to synthesize a variety of different proteins, etc.
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Toxic proteins, membrane proteins
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Small amount expression conditions fumble, solve relative problems professionally, greatly reduce the experimental period, increase the expression quantity
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E. coli Expression System
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High targeted gene expression quantity, low cost, simple culture conditions, product rapidly, strong scalability, simple conversion operation, easy to form disulfide bond
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Prokaryotic proteins, simple eukaryotic proteins
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Expression includes soluble protein, inclusion body, fusion proteins, etc., with wealthy experience and expertise, we can solve a variety of bottlenecks during the protein expression process
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Yeast Expression System
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Cost-effective, low-cost for amplifying medium, simple culture conditions, production rapidly, strong scalability, good choice for secretory protein or intracellular protein expression, secrete proteins efficiently and allow simple purification, extensive post-translational modifications, no endotoxin
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Industrial strain improvement, amplification
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The combination of self-transformed efficient secretion vector and host can achieve the highest quality protein expression to the maximum extent; Patented Biobrick technology can be successfully used to the improvement and optimization of industrial strain
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Insect baculovirus expression system
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Large gene capacity, high efficiency of exogenous gene expression, effective cell fold, moderate scalability, extensive post-translational modifications, glycosylation similar to mammalian cells, is relatively easy enzymatic deglycosylation, no endotoxin
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Virus vaccines, signal proteins, cytokines, kinases, etc.
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Adopt AcNPV-sf9 cells and high5 cells two expression systems, the selectivity of multiple expression systems, multiple hosts, multi-carrier greatly improve the success rate of protein expression
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Mammalian Cell Expression System
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Higher expression levels, moderate scalability, cell suspension culture characteristic can do mass production, effective protein fold, suitable for protein secretion, full post-translational modifications, no endotoxin
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Complex higher eukaryotes proteins
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Adopt the specific combined methods of mammalian cell expression vector and a variety of transfections, optimize expression conditions, improve transfection efficiency, greatly shorten the experimental period, significantly increase the expression quantity
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5. Common Problems of Protein Expression
A. Question about Tag removal, WB identification for Tag antibody, Endotoxin removal, aseptic manufacture process
Answer: Generally, these services such as WB identification of Tag antibody, Endotoxin removal, aseptic manufacture process can apply to all proteins include proteins which are expressed with E.coli system, but the tag removal service depends on the specific proteins, some proteins can remove tag but some can't. The lead time will prolong 2-3 working days accordingly for these services.
B. Question about shipping formats
Answer: The default shipping formats are liquid forms if you don’t have special demands. However, if you have special requirement on the shipping formats, please remark clearly when placing the order.
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CUSABIO team. CUSABIO's Five Expression Systems. https://www.cusabio.com/c-20621.html
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