Overview

Production and purification of proteins with biological activity is essential in drug discovery. Multifunctional units at GenScript secure successful delivery of bioactive proteins based on our state-of-the-art equipment and methodologies. A case study below shows groups of protein production and assay development teaming up deliver enzyme with qualified specific activity, higher than customer expected. After modifying the method of cell-breaking, we increase the final yield of protein of interest with purity of above 90%.

Biological Activity Proteins

Fig. A: SDS-PAGE analysis of the fractions upon the IEX chromatography

Lane 1-47: Gradient elution fractions

Lane M: Smart Broad-Range Protein Standard (GenScript, Cat. No. MM0906)

Lane S: The supernatant after homogenizer with high pressure

Lane ft: Flow through

Biological Activity Proteins

Fig. B: Enzyme assay monitoring the fractionation by DEAE Sepharose

Biological Activity Proteins

Fig. C: Fractions pooled based on activity analysis for further ADP Sepharose affinity purification

Fig. D: SDS-PAGE analysis of the purified protein

Lane M: Smart Broad-Range Protein Standard (GenScript, Cat. No. MM0906)
Lane S: The supernatant after homogenizer with high pressure
Lane ft: Flow through

Large-scale Endotoxin Free Protein Production

GenScript's protein team has extensive experience in Bacterial Expression And Purification, protein refolding, solubility enhancement, endotoxin removal, and large-scale production.

In a recent case, we have obtained 108 mg A fusion protein with >90% purity from 20 L E. coli expression and two-step purification. The concentration is 4.02 mg/ml and the endotoxin level is lower than 0.8 EU/µg.

SDS-PAGE results

Fig. 1 SDS-PAGE analysis of the expression of A fusion protein

Fig. 2 SDS-PAGE analysis of the first step purification of A fusion protein

Lane M: Smart Broad-Range Protein Standard (Genscript, Cat.No. MM0906) Lane 1: Supernatant fraction Lane 3: Wash
Lane 2: Flow through fraction Lane 4–5: Elution
Lane 1: Lysate of cells without induced Lane M: Smart Broad-Range Protein Standard (Genscript, Cat.No. MM0906)
Lane 2–4: Lysate of cells induced
Enzyme assay results

Fig.3 SDS-PAGE analysis of the second step purification of A fusion protein

Fig. 4 SDS-PAGE analysis of the purity of A fusion protein

Lane 1: A fusion protein
Lane M: Smart Broad-Range Protein Standard (Genscript, Cat.No. MM0906)
Lane 1: Specimen fraction Lane 2–9: Elution
Lane M: Smart Broad-Range Protein Standard (Genscript, Cat.No. MM0906)
  • GenSmart™ Codon Optimization Tool Technology

    Proven increase in protein expression through optimizing a variety of parameters

    codon optimization case study
    codon optimization case study

    Fig. 1. GenScript OptimumGene™ codon optimization genes increased the yield of expression (8 out of 10 genes) and the degree of solubility in some cases (6 out of 10 genes) compared to the native genes.

    Reference: Burgess-Brown NA, Sharma S, Sobott F, Loenarz C, Oppermann U, Gileadi O. Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene study. Protein Expr Purif. May 2008; 59(1): 94-102

    Significant Increase Drug Target Proteins Expression Level in E.coli.

    Figures below manifested the effectiveness of the OptimumGene™ technology in increasing drug target proteins expression levels for two different genes expressed in E.coli.

    protein activity analysis results
    SDS-PAGE analysis protein expression

    Fig. 2. GenScript's OptimumGene™ codon optimization delivered 10 times higher expression level for protein α compared to non-optimized native gene sequence. The expression level was 3 times more than that from Competitor GA's optimization method.

    SDS-PAGE results of protein B
    expression results of protein B

    Fig. 3. GenScript's OptimumGene™ codon optimization delivered 20 times higher expression level for protein β compared to non-optimized native gene sequence. The expression level was 13 times more than that from Competitor GA's optimization method.

  • Co-expression of Protein Complexes

    Purification of macromolecular complex subunits in large quantities and assembling them into functional entities are vital steps for successfully determining the structure and characterizing the functions of macromolecular complexes. Despite the fact that cases of isolation and structure determination of endogenous complexes have been demonstrated, great efforts still lie ahead to make this technology easily accessible. Recent endeavor on this subject suggests that co-expression of subunits within hosts of E.coli and insect cells, even for high-throughput projects, is becoming a promising solution.

    GenScript has successfully co-expressed 3 subunits of A protein in E. coli system:

    Co-expression of protein complexes

    Fig. A protein with His-tag was analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining
    Lane M: Smart Broad-Range Protein Standard (GenScript, Cat. No. MM0906)
    Lane 1: A-His-αβγ target protein with His-tag

  • BacuVance Baculovirus Expression System

    1. Project evaluation and high secretory expression level (6mg/L)

    Customer's initial request was to synthesize the gene in question and to clone it into our BV vector for intracellular expression. The expected yield was 0.1-1 mg of tagged protein with over 70% purity. Based on a thorough sequence analysis, we designed a new strategy, which was approved by the customer, touse the protein's native signal peptide to drive secretion. In the end, we obtained 3 mg of protein with a purity level over 80% from 0.5 L SF9 CM. The protein's identity was further confirmed by MALDI-TOF.

    Baculovirus Expression System

    Fig. A: SDS-PAGE Analysis of target protein purified by SP column
    Lane 1-8: Eluted fractions with 1 M NaCl in 20 mM PB, pH 5.0
    Lane M: Protein Marker

    2. Tag-free protein purification

    After protein expression, the conditioned medium was dialyzed against buffer, and loaded onto SP column. The predicted size of the target protein was 78 kDa. The SDS-PAGE results showed that proteins of the same size as the target had been obtained in lanes 12, 13, 14.

    Baculovirus Protein Purification

    Fig. B: SDS-PAGE Analysis of target protein purified by SP column
    Lane 1: The condition medium
    Lane 2-14: Eluted fractions with gradient concentration of NaCl from 0 to 500 mM in 20 mM PB, pH 5.0
    Lane M: Protein Marker

  • Mammalian Expression System

    GenScript recommends its mammalian expression services for cases in which the folding of the proteins is crucial to the customer's intended application and cases in which the internal machinery of E. coli is unable to express the protein with desired conformation and modification. Our proprietary 293 and CHO suspension system platform promotes fast production and delivery of recombinant proteins and monoclonal antibodies up to grams level.

    1. Monoclonal Antibody Production

    SDS-PAGE analysis and IEF analysis
    Fig. 1-1 SDS-PAGE analysis of A Antibody expressed in our mammalian expression system
    Lane 1 and 2: reduced; Lane 3 and 4: non-reduced
    Fig. 1-2 IEF analysis of A Antibody expressed in our mammalian expression system

    2. Recombinant Protein Expression

    Recombinant Protein Expression
    Fig. 2-1 SDS-PAGE analysis of A Protein expressed in our mammalian expression system
    Lane 1 and 2: reduced; Lane 3 and 4: non-reduced
    Fig. 2-2 Western Blot analysis of A Protein expressed in our mammalian expression system

    3. Membrane Protein

    Membrane Protein

    Fig. 3 Indirect Immunofluorescence detection of cell membrane protein expressed in our mammalian expression system

  • Newly launched TurboCHO™ antibody expression

    1. High expression titers of multiple isotypes in different TurboCHO™ platforms

    image-1
    image-2
    image-3

    2.Bispecific Antibody Production, Titer: 374 mg/L with Purity > 99%

    image-4
    image-5
    image-6

Quotations and Ordering

  • To request a quotation, please download and complete the Quotation/Order Form, and send it to us by email or fax.
  • To order, please download and complete the Quotation/Order Form and send it to us by email or fax with a formal PO (Purchase Order) or credit card information. You can also submit PO/credit card information by phone or via our secure online messaging system.
  • If submitting samples, please mail them together with a hard copy of the completed Quotation/Order Form to: Recombinant Protein Services, GenScript, 860 Centennial Ave., Piscataway, NJ 08854, US.
  • For questions about recombinant protein expression, or to inquire about the status of your order contact us by email, phone, fax or via our secure online messaging system.

Our customer service representatives are available 24 hours a day, Monday through Friday to assist you.

REQUEST A QUOTE

feedback

Do you like the current new website?

Hate

Dislike

Neutral

Like

Love

*