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Case Studies

Protein with Biological Activity

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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%.

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

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

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/ug.

• OptimumGene™ Codon Optimization Technology     See more  

  • Proven increase in protein expression through optimizing a variety of parameters

    
    

    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.

    

    

    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.

    

    

    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     See more  

  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:

  

  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
• BacPower™ Bacterial Expression System     See more  

  1. Protein refolding

  Overproduction of proteins in E.coli can cause the formation of inactive, misfolded and insoluble protein aggregates. GenScript provides proprietary refolding technology to solve this problem. A case study shows that over 95% of the inclusion body is solublized and refolded. The purity of the refolded protein is more than 85%. The refolded protein is delivered in customized buffer.

  

  Fig. A: Refolded protein of sample was analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining
  Lane M: Smart Protein Standard (Middle-Range) (GenScript, Cat. No. MM0900)
  Lane 1: Refolded protein of sample

  2. Tag removal

  Presence of epitope tag in recombinant proteins may result in changes in protein structure, toxicity, and loss of bioactivity. To avoid these problems, tag removal after protein expression is one of additional requests from some customers. Shown below is protein that is cleaved by SUMO protease and successfully isolated. About 80% tag free target protein with purity of >90% is recovered.

  

  SDS-PAGE checking purification of protein after tag cleavage by SUMO protease
  Fig. B:
  Lane M: Smart Protein Standard (Middle-Range) (GenScript, Cat.No. MM0900)
  Lane 1: Refolded fusion protein with SUMO-tag
  Fig. C:
  Lane M: Smart Protein Standard (Middle-Range) (GenScript, Cat.No. MM0900)
  Lane 1: Recovered protein after 1st incubation with Ni-resin
  Lane 2: Recovered protein after 2nd incubation with Ni-resin
  Lane 3: Elute with 300 mM imidazole from Ni-resin of 2nd incubation
• YeastHIGH™ Yeast Expression System      See more  

  1. Protein expression in yeast

  GenScript's proprietary YeastHIGH™ expression system and in-house YeastFast technology delivers unparalleled advantages in secretory expression. Pictured below is an enzyme expression performed using our YeastHIGH™ expression technology. After one step purification by SP-Sepharose, we find tag-free proteins at an expression level of about 20 mg/L and at purity level of over 90%. The target protein is further identified by MALDI-TOF.

  

  Fig. A: Expression and secretion of target protein from P. pastoris
  Lane 1: Conventinal yeast expression technology
  Lane 2: GenScript YeastHIGH™ technology
  Lane 3: Negative control
  Lane M: Smart Protein Standard (Middle-Range) (GenScript, Cat. No. MM0900)
  Fig. B: Purification of the target protein by SP-Sepharose
  Lane  M: Smart Protein Standard (Middle-Range) (GenScript, Cat. No. MM0900)

  
  2. Signal peptides

  Shown below is the expression of a protease with signal peptide. There was no secretion expression of target protein with Saccharomyces cerevisiae α factor prepro peptide. After changing the peptide sequence for secretion from α factor prepro to the native signal peptide of the protease itself, the target protein was expressed and secreted successfully.

  

  Fig. C: Western blot analysis of target protein expression with native signal peptide
  Lane 1-5: Induced for 24h, 48h, 72h, 84h, 120h
  Lane M: Protein Marker
  Fig. D: Western blot analysis of target protein expression with α factor prepro peptide
  Lane 1-4: Induced for 24h, 48h, 72h, 84h
  Lane M: Protein Marker
• BacuVance™ Baculovirus Expression System     See more  

  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.

  

  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.

  

  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     See more  

  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

  

  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

  

  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

  

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

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.

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