Case Studies

Case studies

Recombinant protein production is rather empirical and relies largely on experience. Scientists at Novoprotein have obtained profound knowledge and skills in strain/vector selection, buffer composition, pH stringency, and other parameters that determine a successful protein expression and purification. Click on the case studies to see how your projects will be accomplished and how the quality is guaranteed. Call our service specialists at (908) 219-7041 should you have any questions.

Case studies : Polyclonal antibody

  • Strategy design

    • Choose peptide antigen according to intended application of antibody and protein characters
  • Work flow:

    • Peptide design and synthesis
    • Conjugation of 5mg peptide to KLH via terminal Cysteine
    • Immunization of 2 rabbits using conjugated peptide
    • Boosts for 5 times and anti-sera titer to 1:8000
    • Collection of 100ml final immune bleeds
  • Deliverables:

    • Polyclonal antibody with titer greater than 1:2,048,000
  • 1. Peptide antigen design, synthesis and QC

    Fig. 1 HPLC result of synthesized peptide antigen, purity >80%
  • 2. Antigen affinity purification of antibody

    Fig. 2 SDS-PAGE analysis of antibody after antigen affinity purification
  • 3. ELISA Titer Test of Boost #5 Anti-serum and Final Product

    Fig. 3
  • 4. Immunofluorescence detection using our polyclonal antibody

    Fig. 4

For more information about our polyclonal antibody service, or need help to decide whether peptide or protein antigen is suitable for you, contact us at sales@novoprotein.com or visit our Technical support page. In addition, we also offer monoclonal antibody production and antibody fragmentation services. Want to know how other scientists speak of us? Check out our References.

YOUR CONCERNS

Will I get what I am promised?

Our counter-contract ensures your terms of expectation are met.

I feel like losing control of data generation!

Our service team keeps tight monitoring and tracking over each step where data are generated.

Case studies : Recombinant monoclonal antibody

  • Strategy design

    • Genes encoding heavy and light chains of the antibody were constructed into Novoprotein high expression plasmids.
    • Confirm by sequencing.
    • Small scale expression and purification.
    • Sample sent to customer and large scale production initiated.
  • Deliverables:

    • 100mg antibody was produced with concentration >2mg/ml, purity>90% as estimated on SDS-page.
  • 1. Small scale expression

    Fig. 1 SDS-PAGE analysis of small scale expression.

    1-3: 72-120 hour expression; 4: expression control GFP
  • 2. Purification of antibody by protein A column

    Fig. 2 SDS-PAGE analysis of antibody after protein A purification.

    1: Loading sample; 2: Flow-through; 3-9:Elutions 1-6
  • 3. Customer data: Novoprotein Antibody binding property on purified antigen and cells

    Fig. 3 Compare to Dr. XX lab’s antibody, the antibody produced by Novo protein has the same cell binging affinity on Hep3B cells and G1 cells.
  • 4. Large scale production of antibody

    Fig. 4 SDS-PAGE analysis of antibody after purification by protein A column

    1: Loading sample(7L)

    2: Flow-through

    3: Elution by 0.1MGlycine pH 3.0

    4: Elution by 0.1MGlycine pH 3.0

    5: Lane4 sample (non-reduced)

    6: Elution by 0.1MGlycine pH 3.0
  • 5. Final product

    Fig. 5 Final product.

    A: reduced

    B: non-reduced

    Concentration:2.48mg/ml

    Amount:101.6mg

For more information about our monoclonal antibody service, contact us at sales@novoprotein.com or visit our Technical support page. In addition, we also offer monoclonal antibody production and antibody fragmentation services. Want to know how other scientists speak of us? Check out our References.

YOUR CONCERNS

Will I get what I am promised?

Our counter-contract ensures your terms of expectation are met.

I feel like losing control of data generation!

Our service team keeps tight monitoring and tracking over each step where data are generated.

Case studies : Isotope labeling service

Case study - Production of 15N and 13C Labelled Protein in E. coli System

  • Workflow:

    • 10 liters of high expression clone was grown in 15N and 13C labeled minimal medium and induced with IPTG
    • Target protein was expressed as inclusion bodies
    • Target protein was refolded and the unwanted pro-peptide removed
    • Mature protein was purified by IEX columns
  • Deliverables:

    15N and 13C labeled target protein

  • 1. Purification of refolded protein

  • Fig. 1 SDS-PAGE gel analysis of target protein after refolding and pro-peptide removal

    A: Purified Protein (reduced)

    B: Purified Protein (non-reduced)
  • 2. MS Assay Result of Final Product

    Fig. 2 MS analysis of the final product

    MW of the test sample: 13031.6Da

    MW of unlabeled target protein: 12374.2 Da

    MW of 15N and 13C labeled target protein: 13070.2 Da
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We also offer post-purification services including process development, protein characterization, protein crystallization, peptide synthesis, and mammalian stable cell line generation. For information about our protein expression and purification services, please visit our protein expression and purification page or contact us at sales@novoprotein.com. Want to know how other scientists speak of us? Check out our References.

YOUR CONCERNS

Will I get what I am promised?

Our counter-contract ensures your terms of expectation are met.

I feel like losing control of data generation!

Our service team keeps tight monitoring and tracking over each step where data are generated.

Case studies : Large Scale Production / Process Development

Case study – Process Development and Pre-clinic Study of Protein Drug Candidate

  • Workflow:

    • The target gene was codon optimized and synthesized.
    • The gene was subcloned into pPICZa vector.
    • Transformation of plasmid into high-efficiency X33 P. pastoris cell.
    • Antibiotic selection of high expression strain.
    • Expression of target protein in 50L fermentor in GMP plant.
    • The target protein was purified by Blue, HIC and SEC resin.
  • Deliverables:

    Protein product tested in in vitro bioactivity, pharmacokinetics, and half life in monkey

  • 1. Target Protein Production in P. pastoris System

    Fig. 1 SDS-PAGE analysis of target protein after purification

    A: Purified protein (non-reduced)

    B: Purified protein (reduced)

    MK: Molecular weight standards
  • 2. Characterization of target protein product

    HPLC
    Mass Spectrum
  • 3. Pre-clinic Study of Protein Product

    In vitro bioactivity
  •  

    Pharmacokinetics in monkeys
  •  

    Half life in monkey

We also offer post-purification services including isotope labeling, protein characterization, protein crystallization, peptide synthesis, and mammalian stable cell line generation. For information about our protein expression and purification services, please visit our protein expression and purification page or contact us at sales@novoprotein.com. Want to know how other scientists speak of us? Check out our References.

YOUR CONCERNS

Will I get what I am promised?

Our counter-contract ensures your terms of expectation are met.

I feel like losing control of data generation!

Our service team keeps tight monitoring and tracking over each step where data are generated.

Case studies : Protein characterization

Case study 1 - Characterization and Bioactivity Test of Cytokine

  • Workflow:

    • The target cytokine gene was synthesized and subcloned into pET30a vector.
    • Plasmid was transformed into high-efficiency BL-21 E. coli cell.
    • High expression E. coli strain was selected by antibiotics
    • Large-scale culture to express target cytokine.
    • The cytokine was purified by IEX and SEC resin.
  • Deliverables:

    Bioactive cytokine

  • 1. Production of Cytokine in E. coli System

  • Fig. 1 SDS-PAGE analysis of cytokine after purification

    A: Purified cytokine (non-reduced)

    B: Purified cytokine (reduced)

    MK: Molecular weight standards
  • 2. Characterization of cytokine product

    Fig. 2 Characterization of cytokine product by mass spectrum
  •  

    Sequencing verification by MS/MS assay
  • 3. Bioactivity Assay of final cytokine product

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    Fig.3 Bioactivity assay showing Novoprotein cytokine with similar bioactivity as commercial product in market.

Case study 2 – Endotoxin level control

Endotoxin level (Eu/mg) Protein concentration(ug/ml) Protein concentration(ug/ml) Dilution times LAL result (positive+, negative -)
1000 0.5 1000 -
1000 0.5 1000 -
500 1 500 -
500 1 500 -
100 5 100 -
100 5 100 -
10 50 50 -
10 50 50 -
5 25 10 -
5 25 10 -
LAL(Limulusamebocytelysate) controlled to 5EU/mg

We also offer post-purification services including isotope labeling, process development, protein crystallization, peptide synthesis, and mammalian stable cell line generation. For information about our protein expression and purification services, please visit our protein expression and purification page or contact us at sales@novoprotein.com. Want to know how other scientists speak of us? Check out our References.

YOUR CONCERNS

Will I get what I am promised?

Our counter-contract ensures your terms of expectation are met.

I feel like losing control of data generation!

Our service team keeps tight monitoring and tracking over each step where data are generated.

Case studies : Mammalian stable cell line generation

Case study – Generation of Mammalian Stable Cell Line

  • Workflow:

    • Protein A and Protein B were constructed into expression vector.
    • Co-Transfect into CHO cells..
    • Stable lines are selected with G-418 and MTX, then target gene is amplified with gradually increased MTX concentration.
    • Protein A and Protein B expression was detected with corresponding antibodies.
    • Gene function was determined by phospho-AKT antibody at the present of NRG.
  • Deliverables:

    Bioactive cytokine

  • 1. Target Protein Production in P. pastoris System

    Fig.1 Detection of protein A and protein B expression
  • 2. Characterization of target protein product

    Fig.2 Determination of gene function with regards to its dose-response to NRG

We also offer post-purification services including isotope labeling, process development, protein characterization, protein crystallization, and peptide synthesis. For information about our protein expression and purification services, please visit our protein expression and purification page or contact us at sales@novoprotein.com. Want to know how other scientists speak of us? Check out our References.

YOUR CONCERNS

Will I get what I am promised?

Our counter-contract ensures your terms of expectation are met.

I feel like losing control of data generation!

Our service team keeps tight monitoring and tracking over each step where data are generated.

Case studies : E. coli expression system

  • Protein property:

    • Secreted cytokine
    • Expressed as pro-peptide (33kD), processed to mature form (14kD)
  • Challenges:

    Both pro-peptide and mature forms are expressed in inclusion body (IB)

  • Strategy:

    • Pro-peptide modification to facilitate the refolding
    • Solubilize and refold pro-peptide
    • Remove pro-peptide sequence
  • Deliverables:

    Refolded bioactive mature peptide above 95% purity

  • 1.Expression strain screening

    Fig.1 SDS-PAGE analysis of pro-peptide expression.

    Lane2: After inducing

    Lane3: Before inducing
  • 2.Inclusion body solubilization and pro-peptide refolding

    Fig.2 Solubilization and refolding of inclusion body in NP proprietary buffers.

    IB: Inclusion body

    R: Soluble and folded pro-peptide, Reduced

    NR: Soluble and folded pro-peptide, Non-Reduced
  • 3.Purification of refolded pro-peptide

  • Fig.3 IEX purification (SP-HP) of refolded pro-peptide

    S: Sample

    FT: Flow Through

    E1: Elution1

    E2: Elution2
  • 4.Mature peptide processing

    Fig.4 Pro-peptide processed into mature form with NP proprietary reagents and buffer.

    pp: Pro-peptide

    mp(R): Mature peptide (reduced)

    mp(NR): Mature peptide (non-reduced)
  • 5.Final product: refolded bioactive mature peptide with purity above 95% (Mass Spec confirmed)

    Fig.5 Final product analyzed by SDS-PAGE.

    Fraction1: 0.24mg/ml(A280)

    Fraction2: 0.42mg/ml(A280)

    Fraction3: 0.86mg/ml(A280)

For more information about our E. coli expression service, contact us at sales@novoprotein.com or visit our Service page. In addition, we also offer other protein expression services including yeast protein expression services, baculovirus expression services,mammalian expression service. To know how other scientists speak of us, check out our References.

YOUR CONCERNS

Will I get what I am promised?

Our counter-contract ensures your terms of expectation are met.

I feel like losing control of data generation!

Our service team keeps tight monitoring and tracking over each step where data are generated.

Case studies : Insect expression system

  • Challenge:

    • Protein difficult to purify
  • Strategy:

    • Two-step purification - Heparin & SP columns
    • Step gradient & linear gradient
  • Deliverables:

    • refolded bioactive mature peptide above 95% purity
  • 1. Concentration and 1st Purification – His Affinity Column (20ml)

    • 6 liters cell culture supernatant concentrated by VIVA200 (COMW 30k) ultrafilter to 1.2L.
    • Concentrated solution was purified by 10ml His affinity column. Protein bonded to His column was eluted by step imidazole gradient.
    • Elute fraction 3 was pooled for 2nd purification


    A: Cell culture supernatant (before dialysis)

    B: Cell culture supernatant (after dialysis)

    D: Concentrated solution

    E: Flow-through of His column

    F-H: Elute fractions 1-3

  • 2. 2nd Purification – His HP Column (5ml)

    • Protein bound to His HP column was eluted by three step imidazole gradient
    • Elute fraction 3 was pooled as final product

    A:Loading sample; B:Flow-through; C-E:Fraction 1-3;

    F:Tail of fraction 3

  • 3. Final product

For more information about our yeast expression service, contact us at sales@novoprotein.com or visit our Service page. In addition, we also offer other protein expression services including bacteria protein expression service, yeast expression services,mammalian expression service. To know how other scientists speak of us, check out our References.

YOUR CONCERNS

Will I get what I am promised?

Our counter-contract ensures your terms of expectation are met.

I feel like losing control of data generation!

Our service team keeps tight monitoring and tracking over each step where data are generated.

Case studies : Yeast Protein Expression System

  • Challenge:

    • Protein difficult to purify
  • Strategy:

    • Two-step purification - Heparin & SP columns
    • Step gradient & linear gradient
  • Deliverables:

    • refolded bioactive mature peptide above 95% purity
  • 1. Expression strain screening: 3 clones with highest recombinant protein expression rate were selected

    Fig. 1 SDS-PAGE analysis of target protein expression.

    A: Negative control (empty vector, reduced)

    B: Negative control (empty vector, non-reduced)

    1-3: Clones 1-3 after inducing (R: reduced; NR: non-reduced)
  • 2. Purification using Heparin Column – step ion gradient

    Fig. 2 Heparin column purification under step ion gradients

    A: Cell culture supernatant; B: Flow-through

    1-5: Eluted fractions under ion gradients (reduced)

    2-10: Eluted fractions under ion gradients (non-reduced)

    Eluted fractions 2-4 were pooled for 2nd purification
  • 3. 2nd purification using SP HP column – linear ion gradient

    Fig. 3 SP column purification under linear ion gradients

    A: Cell culture supernatant; B: Flow-through

    1-14: Eluted fraction 1 – 14

    Eluted fractions 3-10 were pooled as final product
  • 4. Final product

    Fig. 4 Final product

    R: reduced;

    NR: non-reduced

For more information about our yeast expression service, contact us at sales@novoprotein.com or visit our Service page. In addition, we also offer other protein expression services including bacteria protein expression service, baculovirus expression services,mammalian expression service. To know how other scientists speak of us, check out our References.

YOUR CONCERNS

Will I get what I am promised?

Our counter-contract ensures your terms of expectation are met.

I feel like losing control of data generation!

Our service team keeps tight monitoring and tracking over each step where data are generated.

Case studies : Mammailian cell expression system

  • Protein properties:

    • secreted protein, with two 14kDa subunits
  • Challenges:

    • purify non-covalently linked, no tagged heterodimers
  • Strategy:

    • Clone each subunit to an individual construct
    • Co-transfection with two subunit constructs at 1:1 ratio
    • Blue Gel affinity: Purify based on binding properties
    • Ion exchange (4 exchanges in total) : Purify based on theoretical PI value
  • Turnaround time:

    • 6 weeks
  • Deliverable:

    • 17mg heterodimer protein above 95% purity
  • 1. Purification I – affinity blue gel: cell culture supernatant loaded

    Fig. 1 SDS-PAGE analysis of protein after affinity purification. Targeted protein bound to blue gel column.

    1: Sample;

    2-3: Flow-through;

    4: Elution (reduced);

    8: Elution (Non-Reduced)
  • 2. Purification II – cation exchange purification (SP-HP): elution of affinity blue gel desalted and loaded

    Fig. 2 SDS-PAGE analysis of protein after SP-HP ion exchange purification. Targeted protein bound at pH 6.0.

    1: sample; 2-3: Flow-through, 4-8: Elutions 1-5
  • 3. Purification III – 1st anion exchange purification (Q-HP): elution of SP-HP desalted and loaded

    Fig. 3 SDS-PAGE analysis of protein after Q-HP ion exchange purification. Targeted protein bound at pH 8.0.

    1: sample; 2-5: Flow-through, 6-8: Elutions 1-3
  • 4. Purification IV – 2nd anion exchange purification (Q-HP): elution of 1st Q-HP desalted and loaded

    Fig. 4 SDS-PAGE analysis of protein after Q-HP ion exchange purification. Targeted protein binding improved yet incomplete at pH 8.8.

    1: sample; 2: Flow-through, 3-6: Elutions 1-4; 9-10: Elutions (non-reduced)
  • 5. Purification V – 3rd anion exchange purification (Q-HP): elution of 2nd Q-HP desalted and loaded

    Fig. 5 SDS-PAGE analysis of protein after Q-HP ion exchange purification. Targeted protein binding is complete at low temperature.

    1: sample; 2: Flow-through, 3-5: Elution (reduced); 9-10: Elution (non-reduced)
  • 6. Final product: Concentration 0.5mg/ml with purity greater than 95%

    Fig. 6 SDS-PAGE analysis of final product.

    1: Reduced; 3: Non-reduced (heterodimer)

For more information about our mammalian expression service, contact us at sales@novoprotein.com or visit our Service page. In addition, we also offer other protein expression services including bacteria protein expression service, yeast protein expression services, baculovirus expression services. To know how other scientists speak of us, check out our References.

YOUR CONCERNS

Will I get what I am promised?

Our counter-contract ensures your terms of expectation are met.

I feel like losing control of data generation!

Our service team keeps tight monitoring and tracking over each step where data are generated.