CUSABIO (CSB) Custom Protein Expression

Cosmo Bio USA is pleased to offer a risk-freeCustom Protein Synthesis service using CUSABIO’s Recombinant Protein Expression Platform that features 5 different expression systems:

E. coli Expression System Section
Yeast Expression System Section
Insect Expression System Section
Mammalian Expression System Section
In Vitro E. coli Expression System Section
Custom Protein Expression Order Form

 Why choose CUSABIO?

• Risk-free: No protein, No charge.
• 12 years of experience
• Over 15,000 orders completed
• 99.1% success rate
• Competitive price as low as $535
• 39 kinds of fusion tags meeting different demands
• Secondary AKTA-SEC purification to ensure high purity (>85%)
• Ability to achieve large-scale protein production (10mg, 50mg, 100mg, 200mg)
• Post-purification services available:
• Western blotting (no charge)
• Aliquoting (no charge)
• Endotoxin removal (no charge)
• Aseptic processing (no charge)
• Lyophilization (no charge)
• Desalting (possible extra charge)
• Tag removal (extra charge; no charge if removal is unsuccessful)

Note: This risk-free custom protein service is offered only for proteins up to 800aa. For proteins larger than 800aa charges will accrue according to the different steps required.

 Guide to choosing an expression system

Expression System	System Benefits	Applications
E. coli	High level expression, low cost, simple culture conditions, rapid production, scalability, supports disulfide bond formation	Prokaryotic proteins, simple eukaryotic proteins
Yeast Cell	Cost-effective, low-cost for amplifying medium, simple culture conditions, rapid production, strong scalability, good choice for secretory protein or intracellular protein expression, efficient protein secretion allows simple purification, extensive post-translational modifications, no endotoxin	Industrial and medical strain improvement, amplification
Insect cell Baculovirus	Large gene capacity, high efficiency of exogenous gene expression, effective protein folding, moderate scalability, extensive post-translational modification, mammalian cell-like glycosylation, relatively easy enzymatic de-glycosylation, no endotoxin	Viral vaccines, signal proteins, cytokines, kinases, etc.
Mammalian Cell	Higher expression levels, moderate scalability, cell suspension culture characteristic permits mass production, effective protein folding, suitable for protein secretion, full post-translational modifications, no endotoxin	Functional complex higher eukaryotic proteins
In vitro E. coli	Simple, rapid, high yield and flexible	Toxic proteins, membrane proteins, protein complexes, parallel synthesis of different proteins, etc.

Obtaining a Custom Protein Synthesis quote is a multistep process that begins with filling out this short form [link to https://www.cosmobiousa.com/pages/custom-protein-service]. if you’re not sure of the answers to particular questions, feel free to leave them blank. Following receipt of your order form, we will contact you with either a quotation or follow-up questions. Quotations will include parallel pricing information for each of the expression systems, permitting customers to “comparison shop”. In vitro E. coli expression system quotations will be provided for orders involving transmembrane and cytotoxic proteins or upon request.

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E. coli Expression System (35-45 business days)

 Custom Protein Expression Order Form

Step	Details	Notes	Time Frame
Step 1 (Plasmid construction)	Optimize codon usage and synthesize gene.	Multiple vector optimization to shorten lead time: Optimize multiple vectors at the same time; select the vector with the highest yield.	15-20 business days
	Restriction digest PCR products; ligate to expression vectors (pCold-SUMO, pGEX-4T-1, pET22b-JT, etc.).
	Transform TOP10 E. coil competent cells.
	Select the correct recombinant plasmid.
Step 2 (Transformation and strain screening)	Transform the chosen recombinant plasmid into host cells [BL21(DE3), Rosetta-gami B(DE3)pLysS, C41(DE3)], culture overnight at 37°C.	Multi-condition optimization and multi-host selection: In small-scale tests, optimize induction temperature and IPTG concentration to obtain the most suitable culture conditions. Multiple hosts are transformed at the same time to permit selection of the one with the highest yield.	5 business days
	Select single colonies for small-scale induced expression; detect protein expression by SDS-PAGE; select the best colony.
	Optimize expression conditions.
Step 3 (Target protein expression and purification)	Large-scale (1-10L) expression	Multiple purification methods (optional): Explore different chromatographic conditions including ion exchange, hydrophobic and others using AKTA to determine the optimal purification method.	12-15 business days
	Purify protein
Step 4 (Inclusion body renaturation)	Refold target protein if it accumulates in inclusion bodies.	Diverse refolding methods: Use a variety of buffering conditions to quickly screen for the best one. Achieve protein refolding with purity greater than 90% by dilution renaturation, dialysis renaturation, column chromatography renaturation etc. Solubilization and refolding can be achieved for more than 95% of inclusion bodies.
Step 5 (Additional optional services)	Filter-sterilization: (Note: Lyophilization and Filter-sterilization cannot be performed simultaneously.)	No Charge	2 Business Days
	Lyophilization: (Note: Lyophilization and Filter-sterilization cannot be performed simultaneously.)	No Charge	2 Business Days
	Endotoxin removal: If low endotoxin level is required (e.g., protein will be used in cell culture), we recommend this free service that employs conjugated polymyxin B affinity filter adsorption to remove endotoxin to a guaranteed level within 0.1ng/ug (1EU/ug).	No Charge	2 Business Days
	Tag removal: Not all protein tags can be removed as some proteins will become very unstable after tag removal.	Additional Charge: If removal is unsuccessful, no charge will be applied.	2-3 Business Days
Step 6 (Quality Control)	Test for purity, concentration, etc. QC report will be provided.	Detailed COA report: Detailed product data sheet and COA will be provided for each project.	3-5 business days
	Restriction digest PCR products; ligate to expression vectors (pCold-SUMO, pGEX-4T-1, pET22b-JT, etc.).
	Transform TOP10 E. coil competent cells.
	Select the correct recombinant plasmid.

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Yeast Expression System (35-50 business days)

 Custom Protein Expression Order Form

Step	Details	Notes	Time Frame
Step 1 (Expression vector construction)	Optimize codon usage and synthesize gene.	Multiple vector optimization to shorten lead time: Optimize multiple vectors at the same time; select the vector with the highest yield.	15-20 business days
	Insert PCR amplification products into expression vectors (pPIC9k, pPIC3.5k, pPICZαA, etc.).
	Transform TOP10 E. coil competent cells.
	Select the correct recombinant plasmid.
Step 2 (Transformation and strain screening)	Prepare large quantities of the recombinant plasmid.	High copy screening: Conduct multiple screens for unique markers of different vectors to gradually obtain the highest expressing strain. PickRight Technology:This technology aims to obtain a high expressing strain directly after transformation, shortening screening time by 5-10 business days compared with traditional screening. (This technology is recommended mainly for production of less than 5 mg/L protein.)	10-13 business days
	Linearize the recombinant plasmid.
	Transform recombinant plasmid into GS115, X33, KM71 and other hosts by electroporation.
	Analyze by PCR to identify successful transformants.
	Use geneticin G418, Zeocin and other antibiotics for multiple copies screening to obtain high copy expression.
Step 3 (Small scale test, scale up expression and purification)	Perform small scale expression screening (20-40 strains).	Multiple purification methods (optional): Explore different chromatographic conditions including ion exchange, hydrophobic and others by using AKTA, and then determine the optimal purification method.	7-12 business days/or 15-25 business days if opting for optional Multiple Purification Methods.
	Select optimal strain and optimize expression conditions.
	Scale up culture.
	Purify protein.
Step 4 (Additional optional services)	Filter-sterilization: (Note: Lyophilization and Filter-sterilization cannot be performed simultaneously.)	No Charge	2 Business Days
	Lyophilization: (Note: Lyophilization and Filter-sterilization cannot be performed simultaneously.)	No Charge	2 Business Days
	Tag removal: Not all protein tags can be removed as some proteins will become very unstable after tag removal.	Additional Charge: If removal is unsuccessful, no charge will be applied.	2-3 Business Days
Step 5 (Quality Control)	Test for purity, concentration, etc. QC report is provided.	Detailed COA report: Detailed product data sheet and COA are provided for each project.	3-5 Business Days

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Insect Baculovirus Expression System (35-50 business days)

 Custom Protein Expression Order Form

Step	Details	Notes	Time Frame
Step 1 (Plasmid construction)	Optimize codon usage and synthesize gene.	Vector optimization: In order to improve the success rate of expression and achieve higher yield, we provide recommend using C-terminal fusion tags in addition to conventional N-terminal tags.	15-20 business days
	Insert PCR product into expression vectors (pFastbac1-KHM, pFastBac1-MBP, etc.).
	Transform TOP10 E. coil competent cells.
	Select the correct recombinant plasmid.
Step 2 (Preparation of recombinant Bacmid and high titer virus)	Transform DH10Bac cells; perform PCR analysis to identify correct recombinant Bacmid; produce recombinant bacmid DNA.	Unique suspension transfection method greatly increases the protein expression level and effectively shortens the experimental cycle.	12-15 business days
	Transfect recombinant Bacmid DNA into insect cells to obtain baculovirus; detect expression level by SDS-PAGE; repeat if necessary.
Step 3 (Scale up expression and purification)	Infect insect cells with appropriate baculovirus.	Expression optimization: After optimization, large amounts of protein can be obtained by infecting host cells with low-passage virus.	5-10 business days
	Purify target protein by affinity chromatography, ion exchange, hydrophobic and molecular sieves.
Step 4 (Additional optional services)	Filter-sterilization: (Note: Lyophilization and Filter-sterilization cannot be performed simultaneously.)	No Charge	2 Business Days
	Lyophilization: (Note: Lyophilization and Filter-sterilization cannot be performed simultaneously.)	No Charge	2 Business Days
	Tag removal: Not all protein tags can be removed as some proteins will become very unstable after tag removal.	Additional Charge: If removal is unsuccessful, no charge will be applied.	2-3 Business Days
Step 5 (Quality Control)	Test for purity, concentration, etc. QC report is provided.	Detailed COA report: Detailed product data sheet and COA are provided for each project.	3-5 Business Days

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Mammalian Cell Expression System (35-45 business days)

 Custom Protein Expression Order Form

Step	Details	Notes	Time Frame
Step 1 (Plasmid construction)	Optimize codon usage and synthesize gene.	Multiple vector optimization: To improve the success rate of expression and achieve higher yield, in addition to conventional N-terminal fusion protein expression, we also provide C-terminal fusion tag.	15-20 business days
	Insert PCR amplification products into vectors (pSec series, pCMV series and pcDNA series, e.g.)
	Transform TOP10 E. coil competent cells.
	Select the correct recombinant plasmid.
Step 2 (Small scale expression)	Prepare large quantity of transfection grade recombinant plasmid.	Optimization of transfection conditions: Set different transfection conditions, select the optimal experimental conditions according to the test results.	9-11 business days
	Transiently transfect cells (HEK293, CHO, others).
	Detect expression products.
Step 3 (Scale up expression and purification)	Scale up culture cells and transfect.	Multi-condition expression scheme: According to the protein localization and the best experimental conditions in the small test expression, select different cell lines and different ways of transfection, which can increase the expression quantity, greatly improve the protein expression.	8-9 business days
	Explore different chromatographic conditions, including ion exchange, hydrophobic and others using AKTA to determine optimal purification method.
Step 4 (Additional optional services)	Filter-sterilization: (Note: Lyophilization and Filter-sterilization cannot be performed simultaneously.)	No Charge	2 Business Days
	Lyophilization: (Note: Lyophilization and Filter-sterilization cannot be performed simultaneously.)	No Charge	2 Business Days
	Tag removal: Not all protein tags can be removed as some proteins will become very unstable after tag removal.	Additional Charge: If removal is unsuccessful, no charge will be applied.	2-3 Business Days
Step 5 (Quality Control)	Test for purity, concentration, etc. QC report is provided.	Detailed COA report: Detailed product data sheet and COA are provided for each project.	3-5 Business Days

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In vitro E. coli Expression System (25-35 business days)

 Custom Protein Expression Order Form

Step	Details	Notes	Time Frame
Step 1 (Plasmid construction and preparation)	Optimize codon usage and synthesize gene.	Multi-vector optimization: In order to improve the efficiency of mRNA translation, thereby increasing protein yield, we provide protein expression service using N-terminal peptide optimization in addition to conventional N-terminal fusion protein. The N-terminal peptide contains 6-11 amino acids, it’s the shortest additional amino acid sequence that we have designed.	15-20 business days
	Insert PCR amplification products into pET vectors by restriction enzyme digestion and ligation.
	Prepare large quantity of recombinant plasmid.
Step 2 (Small scale expression)	Multi-condition optimization: SDS-PAGE electrophoresis; determine the optimal reaction condition.	Multi-condition expression scheme: In cell-free expression system, we can express several different proteins simultaneously on the multi-well plate under a variety of different conditions.	7-10 business days (Additional 3 business days for multi-condition purification)
Step 3 (Scale up expression and purification)	Prepare 1-10ml large-scale expression based on results of small-scale expression.	Multi-condition purification scheme (optional): For transmembrane proteins, we provide different detergent purification services to determine the optimum buffer for your transmembrane protein. This purification scheme is most suitable for transmembrane proteins with bioactivity.
	EThe target protein is purified by exploring different chromatographic conditions including ion exchange chromatography, size exclusion chromatography and others by using AKTA, and then determine the optimal purification method.
Step 4 (Additional optional services)	Filter-sterilization: (Note: Lyophilization and Filter-sterilization cannot be performed simultaneously.)	No Charge	2 Business Days
	Lyophilization: (Note: Lyophilization and Filter-sterilization cannot be performed simultaneously.)	No Charge	2 Business Days
	Tag removal: Not all protein tags can be removed as some proteins will become very unstable after tag removal.	Additional Charge: If removal is unsuccessful, no charge will be applied.	2-3 Business Days
Step 5 (Quality Control)	Test for purity, concentration, etc. QC report is provided.	Detailed COA report: Detailed product data sheet and COA are provided for each project.	3-5 Business Days