Case Studies

   

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.

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

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

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

GenScript's proprietary YeastHIGH^TM expression system and in-house YeastFast technology delivers unparalleled advantages in secretory expression. Pictured below is an enzyme expression performed using our YeastHIGH^TM 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 YeastHIGHTM 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)

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

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

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

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