Case Studies

Protein with Biological Activity

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/µg.

	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

	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      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
• 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
• Newly launched TurboCHO™ antibody expression      See more  

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

  

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

  

Quotations and Ordering

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