Custom Services > Protein Expression and Purification
 
Protein Expression and Purification
From Gene to Protein
Starting from de novo gene synthesis, DNA or from almost anywhere a client asks us to begin; Medigene can deliver high quality purified protein in a timely manner.

About Protein Purification
Recombinant proteins are used throughout the whole spectrum of biomedical research. Once you have large quantity of purified protein, you could conduct many downstream experiments, such as enzyme kinetics, functional and structural studies.

There are two main systems for the expression of recombinant protein - prokaryotic and eukaryotic.

Prokaryotic recombinant protein expression systems (E.Coli) have several advantages, including ease of culture, rapid cell growth and relatively simple purification procedures. However, most post-translational modifications are not added by bacteria, and most proteins become insoluble in inclusion bodies and are very difficult to recover as functional proteins.

Eukaryotic systems for the expression of protein include yeast, baculovirus cells, and mammalian cells. Advantages of these systems include high levels of expression, no inclusion bodies, and intact post-translational modifications.

Things to Consider
Sequence Optimization: In highly expressed genes, a narrow set of codons is used and these codons correspond to the more abundant tRNA species. This minimizes the risk of tRNA depletion during translation. In fact, the codons in a gene may be true bottlenecks, especially in cases where foreign genes are expressed in a host in which the usage of codons in highly expressed genes does not resemble the usage of codons in the species from which the foreign gene originates. In such cases, it has been shown that substitution of rare codons in the introduced gene may increase the yield dramatically. In addition, replacement of rare codons might decrease the chance of misincorporation and protect the protein from premature turnover (1).

Expression System: In order to decide which host is most suitable the amount and the degree of purity of the product as well as its biological integrity and potential toxicity should be considered. For example, bacterial expression systems are not suitable if post-translational modification is required to produce a fully functional recombinant product (2).

Expression Vector: Once the host has been selected, many different vectors are available for consideration. However, as for the selection of a suitable host system, the final choice of vector should take into consideration the specific requirements of the application and will, of course, be influenced by the behaviour of the target protein.

Tag: As for the selection of host and vectors, the decision to use which tag must be made according to the needs of the specific application. The two most commonly used tags are glutathione S-transferase (GST tag) and 6 x histidine residues ((His)6 tag).


Protein Expression Systems
Medigene offer all four expression systems* to give you the maximum flexibility.

1. E.Coli Expression System
Escherichia coli (E. coli) is one of the most widely used hosts for the production of heterologous proteins. Its genetics are far better characterized than those of any other microorganism.

Descriptions Deliverables
Guaranteed 15 mg purified soluble protein
Subcloning
Expression optimization and verification
Expression and purification
QC by SDS-PAGE, Western blot*
Tag removal, endotoxin removal service available 		Guaranteed 15 mg purified soluble protein from E.coli expression system

QC data
Guaranteed 5 mg purified soluble protein)
Subcloning
Expression optimization and verification
Expression and purification
QC by SDS-PAGE, Western blot*
Tag removal, endotoxin removal service available 		Guaranteed 5 mg purified soluble protein from E.coli expression system

QC data
Traditional Service
Subcloning
Expression optimization and verification
Expression and purification
QC by SDS-PAGE, Western blot*
Tag removal, endotoxin removal service available 		Purified protein from E.coli expression system

QC data

The guaranteed Packages have the following features:
1. Customer provides the plasmid or the expression strain
2. Size of the target protein is usually <100KDa
3. In some rare cases, in order to facilitate expression, Bio Basic Inc. may try different tags and final protein product may contain such tag
4. The final purified protein is tag-containing
5. Medigene does not guarantee the endotoxin level of purified protein
6. Medigene guarantees the protein to be soluble, but not the bioactivity or functionality. If target protein expresses as inclusion body, refolding will be done to make it soluble.
7. If Medigene failed to deliver the guaranteed amount due to the nature of the protein or other unpredictable circumstances, only a minimum set up fee will apply

An Exciting New Launch 5 mg Intact-Native Protein Expression!
Descriptions Deliverables
Intact Native Protein Expression Package
Subcloning
Expression optimization and verification
Expression and purification
Ultrasonication, Refolding, Purification, Tag Removal
QC by SDS-PAGE, Western blot*		 5 mg of purified protein in its native form
(No Tag, 90- 95% purity, endotoxin <1eu/ug)

QC data
* Certain restrictions apply. Please enquire for further details.

Other E. Coli Expression System Services
Descriptions Descriptions
Lyophilization services Functional preservation and longer shelf life
Fermentation services Scale-up of fermentation and purification process up to 300 liters
cGMP manufacturing services Fully equipped cGMP facility


2. Baculovirus Expression System
The Baculovirus insect cell expression system is widely used for the production of precisely matured, folded and processed recombinant proteins. In contrast to E. coli, insect cells are able to incorporate post-translational modifications. This unique tool usually yields high amounts of the target protein, making it highly cost effective in comparison to other eukaryotic expression systems

Descriptions Deliverables
Subcloning
Generation of recombinant bacmid DNA
Generation of high-titer viral stock
I nfection of insect cells with high-titer virus stocks
QC by SDS-PAGE, Western blot*
Tag removal service available		 High-titer viral stock
Purified soluble protein
QC data


3. Mammalian Expression System
Mammalian cell lines (CHO, HEK 293 etc) are widely used for the production of recombinant glycoproteins (vaccines, enzymes) as well as hormones and immunobiologicals (antibodies, interleukins, lymphokines), as they have the advantage of full post-translational modifications of the expressed protein

Descriptions Deliverables
Transient expression and purification
Stable cell line establishment
QC by SDS-PAGE, Western blot*
Tag removal service available		 Purified protein
QC data
* FREE Western QC data is available for tagged proteins that can be detected by the anti-His antibody. For other tagged proteins, additional charges may apply.
* Yeast expression system is available upon request


Featured Catalogue Products
In addition to the outlined custom protein purification services, Medigene possesses an extensive list of ready-made proteins. This following table includes a sampling of the catalogue protein library (We have a lot more!). If your protein of interest is not on the list, please send an email to info@mdgsb.com to enquire.

Interleukins Growth Factors Chemokines IFNs Other Specialty Products
Recombinant Human IL-1α Recombinant Human GM-CSF Recombinant Human SDF-1α/CXCL12 Recombinant Human IFN-α1b Recombinant Human TNF-α
Recombinant Human IL-2 Recombinant Human EPO Recombinant Human Lymphotactin/XCL1 Recombinant Human IFN-α2b Recombinant Bovine Enterokinase
Recombinant Murine IL-3 Recombinant Human IGF-1 Recombinant Human RANTES/CCL5 Recombinant Human IFN-γ Recombinant ProteinG
Recombinant Human IL-12 Recombinant Human TPO Recombinant Human MIP-1α/CCL3 Recombinant Human IL-29/IFN-λ1 Recombinant Cys-Protein G
Recombinant Human IL-33 Recombinant Human EGF Recombinant Human GRO-α/CXCL1 Recombinant Rat IFN-γ Recombinant Protein A/G

How to Order
Download and Email: You may download the Protein Order Form and submit your order via e-mail to info@mdgsb.com or ict@mdgsb.com

Protein Expression and Purification Order Form (E.coli) : Download
Protein Expression and Purification Order Form (Insect and Mammalian) : Download

 References
 A. E.Coli Expression System

  Descriptions Deliverables Timeline1
Stage 1
Gene Synthesis
• Obtain cDNA sequence
• Codon optimization if needed
• Gene synthesis
• Synthesized gene
Sequence and size dependent
Stage 2
Subcloning
• Plasmid amplification and purification
• Subclone the gene into expression vector
• Sequence verification
• Purified plasmid
• Plasmid containing bacteria strain
1-2weeks
Stage 3
Transformation
• Plasmid amplification and purification
• Transformation
• 5 clones for protein induction
• High expression E.Coli strain
1-2weeks
Stage 4
Pilot Run
• Pilot run for determination of production yield and other characteristics of the protein
• Progress update on pilot experiment
1-2 weeks
Stage 5
Expression and Purficiation
• Expression condition optimization
• Expression in 1L bacterial culture
• Multiple methods for purification
• Tag removal (optional)
• SDS-PAGE, Western-Blot analysis2
• 3mg - 10 mg of purified protein3
• Purity > 95%
• Final report
2-3 weeks
Stage 6
Additional Services
• Refolding study (optional)
• Endotoxin removal
• HPLC, MS analysis
• Purified protein
• QC report
2-3 weeks
1. Listed is estimated time. Actual time may vary depending on individual project.
2. FREE Western QC data is available for tagged proteins that can be detected by the anti-His antibody. For other tagged proteins, additional charges may apply.
3. Scale up is available upon request. Addition charge will apply.


 B. Baculovirus Expression System

  Descriptions Deliverables Timeline1
Stage 1
Gene Synthesis
• Obtain cDNA sequence
• Codon optimization if needed
• Gene synthesis
• Synthesized gene
Sequence and size dependent
Stage 2
Subcloning
• Plasmid amplification and purification
• Subclone into pFastBac donor vector
• Sequence verification
• Isolation of recombinant plasmid DNA
• Purified plasmid
• Plasmid containing bacteria strain
1-2weeks
Stage 3
Generation of Bacmid DNA
• Selection of recombinant Bacmid DNA containing host strain
• Isolation of recombinant Bacmid DNA
• Bacmid DNA containing strain
• Bacmid DNA
2weeks
Stage 4
Transfection of Insect Cells
• Multiple transfection of Sf-9 to amplify virus
• SDS-PAGE, Western-Blot analysis
• High titer virus stock
3 weeks
Stage 5
Pilot Run
• Pilot run for determination of production yield and other characteristics of the protein
• Progress update on pilot experiment
1-2 weeks
Stage 6
Protein Expression and Purification
• Multiple methods for purification
• Tag removal (optional)
• SDS-PAGE, Western-Blot analysis2
• 0.1 - 2 mg of purified protein3
• >90% purity
• Final report
2-3 weeks
Stage 7
Additional Services
• Endotoxin removal
• HPLC, MS analysis
• Purified protein
• QC report
2 weeks
1. Listed is estimated time. Actual time may vary depending on individual project.
2. FREE Western QC data is available for tagged proteins that can be detected by the anti-His antibody. For other tagged proteins, additional charges may apply.
3. Scale up is available upon request. Addition charge will apply.


 C. Mammalian Expression System

  Descriptions Deliverables Timeline1
Stage 1
Gene Synthesis
• Obtain cDNA sequence
• Codon optimization if needed
• Gene synthesis
• Synthesized gene
Sequence and size dependent
Stage 2
Subcloning
• Plasmid amplification and purification
• Subclone into eukaryotic expression vector
• Sequence verification
• Isolation of recombinant plasmid DNA
• Purified plasmid
• Plasmid containing bacteria strain
1-2weeks
Stage 3
Pilot Run
• Pilot run for determination of production yield and other characteristics of the protein
• SDS-PAGE, Western-Blot analysis
• Progress update on pilot experiment
2 weeks
Stage 4
Protein Expression and Purification
• Protein production using transient gene expression
• Multiple methods for purification
• Tag removal (optional)
• SDS-PAGE, Western-Blot analysis
• 0.1 - 2 mg of purified protein3
• >90% purity
2-3 weeks
Stage 5
Additional Services
• Endotoxin removal
• HPLC, MS analysis
• Purified protein
2-3 weeks
Stage 6
Stable Cell Line Development
• MTX selection
• SDS-PAGE, Western-Blot analysis2
• Stable cell line
6-8 weeks
1. Listed is estimated time. Actual time may vary depending on individual project.
2. FREE Western QC data is available for tagged proteins that can be detected by the anti-His antibody. For other tagged proteins, additional charges may apply.
3. Scale up is available upon request. Addition charge will apply.
 
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