Custom Protein Expression

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Introduction

Cosmogenetech Inc., an industry leader in gene, peptide synthesis, is pleased to launch the new protein expression and purification service. Cosmogenetech Inc. has the necessary know- how to deliver the best possible esults to our valued customers.

We have the capacity to take your project directly from gene synthesis to protein expression, refolding and purification.

At Cosmogenetech Inc., we are proud of our ability to optimize protein expression, maximize product yield, and ensure purity to serve every aspect of your research needs.

From Gene to Protein

Starting from de novo gene synthesis, cDNA or from almost anywhere a client asks us to begin; our scientists 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.

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.

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).

E.coil

expression service

Step Description Timeline Price

Step 1 Pretest Optimization of expression condition 1 week 500,000 Generation and identification of bacterial colonies expressing target proteins

- Small scale expression은 large scale expression을 하기 전에 반드시 해야 할 단계로,

Expression optimizaion(Induction Time, IPTG concentration, Growth temperature, Bacterial strains, Protein solubility) 하는 단계입니다.

Step 2 Western blotting Expression protein test 1 week 200,000 Pretest의 SDS-PAGE 분석을 통하여 Expression 조건이 결정되면,

목적 단백질에 tagging된 antibody(His, GST, MBP 등)를 이용하여 발현된 단백질을 확인하는 단계입니다

(anti-His or GST 외의 antibody 확인 요청 시 Ab 제공 필요)

Step 3 Purification Protein Purification 1 week 300,000

1. One liter of bacterial culture will be induced with IPTG and harvested for protein purification 2. Protein purification using

- Ni-NTA (for 6xHIS tagged proteins)

- Glutathione (for GST-tagged proteins) beads - MBP resin at either native or denatured conditions

3. Non-tagging protein purification

Total Protein Expression 3 weeks 1,000,000

Protein Expression(10건) 5 weeks 20% DC

Addional service Taq removal 1 week 300,000

◘ COSMOGENETECH

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E.coil

expression service

Description Deliverables Price

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 5mg purified

soluble protein from E.coli expression system QC data

From 3,244,000

Guaranteed 15 mg purified soluble protein

Subcloning

Guaranteed 15mg purified

soluble protein from E.coli expression system QC data

From 4,325,000

Traditional Service

Subcloning

Purified protein from E.coli expression system QC data

Please inquire

a. The guaranteed Packaes (PUR-15MG-

E.coli

& PUR-5MG-

E.coli

) have the following features b. Customer provides the plasmid or the expression strain

c. Size of the target protein is usually < 100KDa

d. In some rare cases, in order to facilitate expression, Bio Basic Inc. may try different rags and final protein product may contain such tag e. The final purified protein is tag-containing

f. Cosmogenetech Inc., does not guarantee the endotoxin level of purified protein

g. Cosmogenetech Ine., 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

h. If Cosmogenetech Inc., failed to deliver the guaranteed amount due to the nature of the protein or other unpredictable circumstance, only a minimum set up fee will apply

An Exciting New Launch 5 mg Intact-Native Protein Expression

Intact Native Protein Expression Package

Subcloning

Ultrasonication, Refolding, Purification, Tag Removal

5mg of purified protein in its native fron

(Notaq, 90-95% purity,endotoxin<1eu/ug)

QC data

From 7,582,000

Other

E. coli

Expression System Services

Lyophilization services Functional preservation and longer shelf life _845,000From Fermentation services Sale-up of fermentation and purification process up to 300 liters _1,183,000From cGMP manufacturing services Fully equipped cGMP facility Please _inquire

Expression pilot study

- Constrution of expressing plasmid containing the cDNA encoding the protein of interest with N-terminus Tag - Small scale transient expression of HEK293 cells

- Expression pilot study, expression level determination by Westhern Blot

QC data Please _inquire

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 posttranslational modifications of the expressed protein

◘ BIOBASIC

Mammalian - Protein production

Transient expression and purification Stable cell line establishment QC by SDS-PAGE, Western blot* Tag removal service available

Purified protein

QC data Please inquire

* 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.

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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

Baculovirus Expression System

Description Deliverables Timeline1 _Price

Stage 1 Gene Synthesis

- Obtain cDNA sequence - Codon optimization if needed

- Gene synthesis Synthesized gene

Sequence and size dependent Visit Gene Synthesis page 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 1-2 weeks

Phase 1 Please inquire 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 2 weeks 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

0.1-2mg of purified protein3

>90% purity

Final report 2-3 weeks _{Please inquire}Phase 2 Stage 7

Additional Services

- Endotoxin removal

- HPLC, MS analysis - Purified protein - QC report 2 weeks

Featured Catalogue Products

In addition to the outlined custom protein purification services, Bio Basic Inc. 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 [email protected] to inquire.

1_{Listed is the typical turnaround time at Bio Basic Inc. 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 oth er tagged proteins, additional charges may apply.

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Antigen manufacture

Antigen Description Lead time Price

Peptide

Peptide design 1 day free

Peptide synthesis 2 weeks inquire

Peptide conjugation (BSA/KLH) 2 day 160,000

Recombinant protein Protein expression 2 weeks inquire

Protein gel elution 1 week 120,000

Antibody manufacture

Immune animals Antigen volume Provides Lead time Price

Mouse (5 heads)

Peptide : 1.3 mg

ELISA identification step-by-step

serum whole serum 10~15 weeks

600,000 Protein : 0.8 mg Strain : 1.0 mg Rat (2 heads) Peptide : 1.2 mg 650,000 Protein : 0.5 mg Strain : 0.8 mg Rabbit (1 head) Peptide : 1.5 mg 700,000 Protein : 1.0 mg Strain : 0.8 mg Rabbit (2 heads) Peptide : 3.0 mg 1,000,000 Protein : 2.0 mg Strain : 1.6 mg Rabbit (3 heads) Peptide : 4.5 mg 1,300,000 Protein : 3.0 mg Strain : 2.4 mg

Antibody purification

Description Volume Lead time Price

Protein A/G affinity purification 10ml 1 week 250,000

PBS buffer exchange 10ml 2 days 250,000

[Cosmogenetech]

Cosmogenetech Inc. produces high quality polyclonal antibody according to peptides and proteins provided by customer or Cosmo itself. Moreover, Cosmo supports one-step service of cloning, protein expression or peptide synthesis based on accumulated skills through cloning business.

[Biobasic]

Introduction

Bio Basic is excited to launch our new Custom Antibody service! We have assembled Custom Antibody (monoclonal and polyclonal) packages that exceed our exacting standards and are sure to meet yours.

Whether you desire antibodies for Western Blot or ImmunoHistochemistry, ELISA or ambitious projects that are highly-specialized to your lab, Bio Basic can help you obtain the results that you need. Our packages are structured give you the freedom to synthesize (from cDNA, peptide or protein) an antibody that will be successful in basic applications or guaranteed to work in your assay of choice.

Our qualified sales staff and experienced scientists will help you choose the Custom Antibody package that suits your project goals. We will keep you informed throughout the generation of custom antibodies.

Features

•ELISA titer guarantee > 1:100,000 (Monoclonal) and > 1:50,000 (Polyclonal) and Dot Blot guarantee •Application-guaranteed monoclonal and polyclonal Ab packages that will always produce the Ab you need •Rapid and constant communication to keep you up-to-date on the status of your project

•All of our shipped antibodies are lyophilized for ease of transport and prevention of degradation

•Industry-leading peptide design and synthesis protocols to ensure your antigens generate optimal immune responses •Highly competitive pricing

Custom Antibody service

◘Polyclonal antibody

Polyclonal antibodies (pAbs) are immunoglobulins that are obtained from different B-lymphocytes. pAbs exist as a heterogeneous mixture of antibodies with differing antigen affinities in serum, and recognize multiple epitopes on any one antigen.

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◘BIOBASIC

1) PolyBasic Standard Peptide Package (Poly-Pep-Stan) 2) PolyBasic Standard Protein Package (Poly-Pro-Stan)

3) PolyBasic Standard Phospho-Specific Package (Poly-Phos-Stan) 4) PolyBasic Guaranteed Phospho-Specific Package (Poly-Phos-Guar)

*모든 antibody제작 과정시 배송료와 peptide 합성 비용은 추가 비용으로 청구됨을 알려드립니다.

PolyBasic Standard Peptide Package (Poly-Pep-Stan)*

Package Name PolyBasic Standard Peptide Package

Package Code Poly-Pep-Stan

Guarantee Bio Basic guarantees to generate antibodies that are Dot Blot-positive against peptide and have ELISA titre > 1:50,000 Provided by Customer Name, species, sequence and NCBI Number for target protein

Service Protocol

1. Peptide design, synthesis and conjugation (additional fees required) 2. Immunize 2 New Zealand Rabbits

3. Serum collection

4. Antigen affinity purification 5. Antibody validation

Project Info Provided ELISA and Dot Blot data against peptide Deliverables 20-50 ml/rabbit antiserum All purified antibody (at least 2 mg)

1 mg peptide

Timeline 6-10 weeks from antigen synthesis to sample delivery

Price Inquire

Payment Policy Client shall provide full payment before project initiation

PolyBasic Standard Protein Package (Poly-Pro-Stan)*

Package Name PolyBasic Standard Protein Package

Package Code Poly-Pep-Stan

Guarantee Bio Basic guarantees to generate antibodies that are Dot Blot-positive against immunogen and have ELISA titre > 1:50,000 Customer to Provide Protein antigen

Service Protocol

1. Qc client's antigen

2. Immunize 2 New Zealand Rabbits 3. Serum collection

4. Protein A affinity purification 5. Antibody validation

Project Info Provided ELISA and Dot Blot data against immunogen Deliverables 20-50 ml/rabbit antiserum _{All purified antibody (at least 20 mg)}

Timeline 4-6 weeks from immunization to sample delivery

Price Inquire

PolyBasic Standard Phospho-Specific Package (Poly-Phos-Stan)*

Package Name PolyBasic Standard Phospho-Specific Package

Package Code Poly-Phos-Stan

Guarantee Bio Basic guarantees to generate antibodies that are Dot Blot-positive against peptide and have ELISA titre > 1:50,000_{and modifide/unmodifide titer ratio > 8} Provided by Customer 1.Name, species, sequence and NCBI Number for target protein_{2. Information of modification site}

Service Protocol

1. Peptide design, synthesis and conjugation, including the modified peptide and unmodifide control peptide (additional fee req.)

4. Antigen affinity purification and cross-reactivity reduction 5. Antibody validation

Project Info Provided ELISA and Dot Blot data against modified and unmodified peptides Deliverables All purified antibody (at least 1 mg)1mg modified peptide

1mg unmodified peptide

Timeline 10-12 weeks from antigen synthesis to sample delivery

Price Inquire

PolyBasic Guaranteed Phospho-Specific Package (Poly-Phos-Guar)*

Package Name PolyBasic Guaranteed Phospho-Specific Package

Package Code Poly-Phos-Guar

Guarantee Bio Basic guarantees to generate antibodies that are Dot Blot-positive against peptide and have ELISA titre > 1:50,000and modifide/unmodifide titer ratio > 8 Bio Basic also guarantees antibody will work in client's experimental system

Provided by Customer 1.Name, species, sequence and NCBI Number for target protein_{2. Information of modification site}

Service Protocol

1. Peptide design, synthesis and conjugation, including the modified peptide and unmodifide control peptide (additional fee req.)

4. Antigen affinity purification and cross-reactivity reduction 5. Antibody validation

Project Info Provided ELISA and Dot Blot data against modified and unmodified peptides Deliverables

First Shipment : Any samples of the client's interest (50ul each) 1mg modified peptide

Second Shipment : All purified antibody (at least 1 mg) Timeline 10-12 weeks from antigen synthesis to sample delivery

Price Inquire

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◘ Monoclonal antibody

Monoclonal antibody service allows us to produce large amounts of pure antibodies in the following way: We can obtain cells that produce antibodies naturally; we also have available a class of cells that can grow continually in cell culture. If we form a hybrid that combines the characteristic of "immortality" with the ability to produce the desired substance, we would have, in effect, a factory to produce antibodies that worked around the clock.

The mAb's were produced based on the ability of a cancer cell's (a myeloma) ability to multiply indefinitely. The stages of producing a mAb are:

" Immunization of the host (usually mice)

" Separation of the immune cells (the plasma cells) " Fusion of the myeloma cell with the plasma cell " Separation of the hwybridoma cells

" Culturing of the cells

" Screening of the cells producing mAb's

◘BIOBASIC

Monoclonal Antibody Packages

MonoBasic Library Package (Mono-Lib-10) MonoBasic Western Blot Package (Mono-WB-1)

MonoBasic Immunofluorescence/Immunocytochemistry Package (Mono-IF-1 / Mono-ICC-1) MonoBasic Immunohistochemistry Package (Mono-IHC-1)

MonoBasic Immunoprecipitation Package (Mono-IP-1) MonoBasic Omni-Application Package (Mono-Omni-1) MonoBasic Phospho-Specific Package (Mono-Phos-1)

Monoclonal Antibody cell line package

많은 수의 (10개) Hybridoma cell line을 원하는 경우!!

MonoBasic Library Package (Mono-Lib-10) ; peptide, protein

모두 가능!!

Package Name Monoclonal Antibody cell line Package

Package Code Mono-Lib-10

Guarantee Bio Basic guarantees to generate at least 10 clones ELISA titre > 1:100,000and positive Dot Blot results against the immunogen

Bio Basic also guarantees that at least 1 clone will work in client's own experiment system Provided by Customer 1.Name, species, sequence and NCBI Number for target proteinOR

2. Protein antigen with < 60% homology to mouse isoform

Service Protocol

1. Antigen disign and production or QC client's antigen 2. Immunize 3 Balb/c mice

3. Fusion and ELISA screening 4. Subcloning (2-4 rounds) 5. Antibody production 6. Antibody validation

Project Info Provided For each cell line:1. Epitope and affinity data (if available) 2. ELISA and Dot Blot data on immunogen Deliverables 10 ELISA-positive hybridoma cell lines

Timeline 10-18 weeks from antigen synthesis/QC to sample delivery

Price Inquire

Payment Policy Client shall pay 50% of price before project initiation_{Client shall pay remaining balance upon project completion}

*Antigen이 Peptide인 경우 Peptide의 합성비용은 추가요금으로 청구되며, 배송료도 추가요금으로 부과됩니다.

MonoBasic IP/IF/ICC/IHC Package

MonoBasic Western Blot Package MonoBasic Library Package (ELISA-guaranteed) MonoBasic Omni-App/ Phospho-Specific Package Application specificity

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Monoclonal Antibody phospho package

Phospho-specific monoclonal antibody제작을 원하는 경우!!

MonoBasic Phospho-Specific Package (Mono-Phos-1) ; Peptide만 가능!! Package Name MonoBasic Phospho-Specific Package

Package Code Mono-Phos-1

Guarantee Bio Basic guarantees to generate at least 1 clone that is Dot Blot-positive against peptide and have ELISA titre > 1:50,000and modified/unmodified titer ratio > 8 Bio Basic also guarantees at least 1 clone will work in client's experiment system

Provided by Customer 1.Name, species, sequence and NCBI Number for target protein_{2. Information of modification site}

Service Protocol

1. Peptide disign, synthesis and conjugation, including the modified peptide and unmodifide control peptide (additional fee req.)

2. Immunize 3 Balb/c mice 3. Fusion and ELISA screening 4. Subcloning (2-4 rounds) 5. Antibody production 6. Protein G affinity purification 7. Antibody validation

Project Info Provided ELISA and Dot Blot data against modified and unmodified peptides Deliverables

First Shipment : Any samples of the client's interest (50ul each) 1mg modified peptide

Second Shipment : 1 hybridoma cell line with 2 mg purified antibody Timeline 16-20 weeks from antigen synthesis to sample delivery

Price Inquire

Payment Policy Client shall pay 50% of price before project initiation_{Client shall pay remaining balance upon confirmation that shipped antibodies work in their experimental system}

*Antigen으로 이용되는 Peptide의 합성 비용은 추가요금으로 청구되며, peptide 합성 소요 기간은 별도로 소요됩니다. 또한 2번의 배송비도 추가요금으로 부과됩니다.

(Monoclonal basic과 phospho package의 경우 1개의 hybridoma cell line을 제공하나, 주문 시 추가로 요구하시면 2개 이하의 hybridoma cell line을 추가 제공 가능함을 알려드립니다 (단, 추가비용이 발생될 수도 있습니다.))

Monoclonal Antibody basic package

ELISA와 Western blot결과까지 원하는 경우!!

MonoBasic Western Blot Package (Mono-WB-1) ; peptide, protein

모두 가능!!

Package Name MonoBasic Western Blot Package

Package Code Mono-WB-1

Guarantee Bio Basic guarantees to generate at least 1 clone that specifically detects the overexpressed antigen by WB_{Bio Basic also guarantees that at least 1 clone detects endogenous antigen in client's WB system} Provided by Customer 1.Name, species, sequence and NCBI Number for target protein_{2. Full length cDNA plasmid}

Service Protocol

1. Antigen disign and production 2. Immunize 3 Balb/c mice 3. Fusion and ELISA screening 4. Subcloning (2-4 rounds) 5. Antibody production 6. Protein G affinity purification 7. Antibody validation

Project Info Provided

For each cell line:

1. Comprehensive validation data on overxpressed antigen, including ELISA and WB data 2. Epitope and affinity data (if available)

3. ELISA and Dot Blot data on immunogen

Deliverables First Shipment : any sanples of client's interest (50ul each)_{Secound Shipment : 1 hybridoma cell line with 2 mg purified antibody} Timeline 14-18 weeks from antigen synthesis to sample delivery

Price Inquire

Payment Policy Client shall pay 30% of price before project initiation_{Client shall pay remaining balance upon confirmation that a qualified clone works in their WB system}

*Antigen이 Peptide인 경우 Peptide의 합성비용은 추가요금으로 청구되며, peptide 합성 소요 기간은 별도로 소요됩니다. 또한 2번의 배송료도 추가요금으로 부과됩니다.