Anti-HER2 scFv Expression in Escherichia coli SHuffle®T7 Express Cells: Effects on Solubility and Biological Activity

Ahmadzadeh, Maryam; Farshdari, Farzaneh; Nematollahi, Leila; Behdani, Mahdi; Mohit, Elham

doi:10.1007/s12033-019-00221-2

Cited by 28 publications

(22 citation statements)

References 46 publications

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“…Recently, different genetically engineered strains like AD494, Origami™ and SHuffle® have been widely used for soluble expression of heterologous proteins ( 24 25 26 ). These modified strains might differ in their intracellular environment, therefore the various soluble yield of recombinant proteins could be achieved for these hosts.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of soluble expression of recombinant granulocyte macrophage stimulating factor (rGM-CSF) by three different E. coli strains

2020

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Background and purpose: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with a wide range of therapeutic applications although expression of GM-CSF in Escherichia coli ( E. coli ) usually leads to formation of insoluble aggregates mostly lack biological activity. The aim of this study was to compare the soluble expression level of GM-CSF in three E. coli strains, BL21 (DE3), SHuffle® T7 and Origami™ 2 (DE3). Experimental approach: The effect of different temperatures and inducer concentrations on soluble expression of GM-CSF was evaluated. The soluble GM-CSF was subjected to endotoxin removal and purification using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, ultrafiltration. The biological activity of produced GM-CSF was evaluated based on its growth promotion effect on TF-1 cell lines by MTT assay method. Findings / Results: A significant improvement of the soluble yield of GM-CSF (about 30% of GM-CSF was expressed as soluble proteins) was observed when protein expression was induced at 30 °C with 0.5 mM isopropyl β- d-1-thiogalactopyranoside (IPTG) in E. coli Shuffle T7. The soluble GM-CSF with a high purity up to 95 % and specific activity of 1.25 × 10 4 IU/μg was obtained. Conclusion and implications: The proposed strategy here can be used to improve the soluble expression of other hard-to-express proteins with similar structural properties ( i.e ., containing disulfide binds or cysteine).

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Section: Discussionmentioning

confidence: 99%

Evaluation of soluble expression of recombinant granulocyte macrophage stimulating factor (rGM-CSF) by three different E. coli strains

2020

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“…PLC/PRF/5 is another hepatoma cell line that expresses Hepatitis virus B surface antigen (https://www.atcc.org/Products/All/CRL-8024.aspx#characteristics). SK‐BR‐3 cell line that is also used in this study as our breast cancer cell model, is an ERBB2 ( HER2 ) positive and metastatic breast cancer cell line which is often employed for assessing the efficiency of anti‐ HER2 therapeutics (Ahmadzadeh et al, 2020; Eloy et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

Validation of common reference genes stability in exosomal mRNA‐isolated from liver and breast cancer cell lines

Gorji-Bahri

Moradtabrizi

Vakhshiteh

et al. 2021

Cell Biology International

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Accurate relative gene expression analysis by reverse transcription‐quantitative polymerase chain reaction relies on the usage of suitable reference genes for data normalization. The RNA content of small extracellular vesicles including exosomes is growingly considered as cancer biomarkers. So, reliable relative quantification of exosomal messenger RNA (mRNA) is essential for cancer diagnosis and prognosis applications. However, suitable reference genes for accurate normalization of a target gene in exosomes derived from cancer cells are not depicted yet. Here, we analyzed the expression and stability of eight well‐known reference genes namely GAPDH, B2M, HPRT1, ACTB, YWHAZ, UBC, RNA18S, and TBP in exosomes‐isolated from the liver (Huh7, HepG2, PLC/PRF/5) and breast (SK‐BR‐3) cancer cell lines using five different algorithms including geNorm, BestKeeper, Delta Ct, NormFinder, and RefFinder. Our results showed that ACTB, TBP, and HPRT1 were not expressed in exosomes‐isolated from studied liver and breast cancer cell lines. The geNorm and BestKeeper algorithms indicated GAPDH and UBC as the most stable candidates. Moreover, Delta Ct and NormFinder algorithms showed YWHAZ as the most stable reference genes. Comprehensive ranking calculated by the RefFinder algorithm also pointed out GAPDH, YWHAZ, and UBC as the first three stable reference genes. Taken together, this study validated the common reference genes stability in exosomal mRNA derived from liver and breast cancer cell lines for the first time. We believe that this study would be the first step in finding more stable reference genes in exosomes that triggers more accurate detection of exosomal biomarkers.

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“…The CyDisCo has been exploited as an efficient route for the production of scFv and Fab fragments derived from known antibodies of different classes and from different organisms (human, mouse and humanized) in the cytoplasm of the KEIO (collection parental K12) E. coli strain in shake flasks [127]. The production of an anti-HER2 scFv with CyDisCo was 251 mg/L using EnPresso B media [54] which is 50.3% higher than the yield obtained without CyDisCo using only EnPresso B (167 mg/L) and 70.5% higher than that obtained in SHuffle using the LB medium (147 mg/L) [109]. Additionally, unlike the ∆trxB/∆gor strains (SHuffle or Origami), CyDisCo, preserving the native cytoplasm environment, results also amenable to large-scale cultivation in chemically defined minimal media as demonstrated by the high-expression yield (139 mg/L) of the scFv of an IgA1 [55].…”

Section: Cytoplasmic Expressionmentioning

confidence: 92%

“…Recently, it has been reported that the expression of an scFv against HER2 derived from Trastuzumab (drug bank number DB00072) in SHuffle at 30 • C resulted in enhanced solubility and a higher expression level of the molecule as compared to its expression in BL21 (DE3) at 37 • C [109][110][111][112].…”

Section: Cytoplasmic Expressionmentioning

confidence: 99%

Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

Sandomenico

Sivaccumar

Ruvo

2020

IJMS

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Antibodies and antibody-derived molecules are continuously developed as both therapeutic agents and key reagents for advanced diagnostic investigations. Their application in these fields has indeed greatly expanded the demand of these molecules and the need for their production in high yield and purity. While full-length antibodies require mammalian expression systems due to the occurrence of functionally and structurally important glycosylations, most antibody fragments and antibody-like molecules are non-glycosylated and can be more conveniently prepared in E. coli-based expression platforms. We propose here an updated survey of the most effective and appropriate methods of preparation of antibody fragments that exploit E. coli as an expression background and review the pros and cons of the different platforms available today. Around 250 references accompany and complete the review together with some lists of the most important new antibody-like molecules that are on the market or are being developed as new biotherapeutics or diagnostic agents.

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Anti-HER2 scFv Expression in Escherichia coli SHuffle®T7 Express Cells: Effects on Solubility and Biological Activity

Cited by 28 publications

References 46 publications

Evaluation of soluble expression of recombinant granulocyte macrophage stimulating factor (rGM-CSF) by three different E. coli strains

Evaluation of soluble expression of recombinant granulocyte macrophage stimulating factor (rGM-CSF) by three different E. coli strains

Validation of common reference genes stability in exosomal mRNA‐isolated from liver and breast cancer cell lines

Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

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