The improvement of anti-HER2 scFv soluble expression in Escherichia coli

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

doi:10.1590/s2175-97902019000317861

Cited by 10 publications

(5 citation statements)

References 26 publications

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“…5 , PDB ID “ 6DN0 ”). The protein was produced and purified from the periplasmic space 41 , 42 , in E. coli BL21 (DE3) co-transformed with pULTRA-pTAFRS and pET22b-HER2-scFv-S9/K42/V15TAG plasmids. A HER2-scFv-K42 pTAF mutant, which had a high expression yield (30 mg/L), was chosen for further characterization; and the mutant and wild-type proteins were confirmed by SDS–PAGE and high-resolution mass spectrometry (Supplementary Figs.…”

Section: Resultsmentioning

confidence: 99%

Noncanonical amino acids as doubly bio-orthogonal handles for one-pot preparation of protein multiconjugates

et al. 2023

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Genetic encoding of noncanonical amino acid (ncAA) for site-specific protein modification has been widely applied for many biological and therapeutic applications. To efficiently prepare homogeneous protein multiconjugates, we design two encodable noncanonical amino acids (ncAAs), 4-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (pTAF) and 3-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (mTAF), containing mutually orthogonal and bioorthogonal azide and tetrazine reaction handles. Recombinant proteins and antibody fragments containing the TAFs can easily be functionalized in one-pot reactions with combinations of commercially available fluorophores, radioisotopes, PEGs, and drugs in a plug-and-play manner to afford protein dual conjugates to assess combinations of tumor diagnosis, image-guided surgery, and targeted therapy in mouse models. Furthermore, we demonstrate that simultaneously incorporating mTAF and a ketone-containing ncAA into one protein via two non-sense codons allows preparation of a site-specific protein triconjugate. Our results demonstrate that TAFs are doubly bio-orthogonal handles for efficient and scalable preparation of homogeneous protein multiconjugates.

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Section: Resultsmentioning

confidence: 99%

Noncanonical amino acids as doubly bio-orthogonal handles for one-pot preparation of protein multiconjugates

et al. 2023

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“…Easy availability in the form of 99 Mo/ 99m Tc generators, commercial availability of 99m Tc labeling kits, suitable half‐life (6.0 h), isomeric decay to a ground state, monochromatic gamma‐ray emission (140.5 keV, 98.5%), highly stable coordination chemistry, and high‐quality SPECT images make technetium‐99m an ideal radioisotope in the field of NM [47, 48]. To our knowledge, in the current study, we radiolabeled the purified his‐tagged anti‐HER2 scFv derived from trastuzumab anti‐HER2 monoclonal antibody [35] with technetium‐99m for the first time by in‐house prepared 99m Tc‐tricarbonyl and evaluated its stability and radiochemical purity.…”

Section: Discussionmentioning

confidence: 99%

“…The anti‐HER2 scFv was expressed in E. coli BL21 (DE3) strain containing pET‐22 (anti‐HER2 scFv) under the optimum condition (Induction with 0.25 mM IPTG at 37°C for 24 h). Due to the presence of His‐tag at the C‐terminal of the scFv, the soluble fraction of scFv was purified via immobilized metal affinity chromatography (IMAC) by Ni‐NTA resin under native condition [35]. The biological activity of the purified anti‐HER2 scFv was confirmed by cell‐based and HER2‐based ELISA [49].…”

Section: Discussionmentioning

confidence: 99%

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Preparation, Characterization, and Radiolabeling of Anti‐HER2 scFv With Technetium Tricarbonyl and Stability Studies

Bozorgchami,

Ahmadzadeh,

Hatamabadi

et al. 2024

Labelled Comp Radiopharmac

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Breast cancer is the most common diagnosed cancer, and the second cause of cancer death among women, worldwide. HER2 overexpression occurred in approximately 15% to 20% of breast cancers. Invasive biopsy method has been used for detection of HER2 overexpression. HER2‐targeted imaging via an appropriate radionuclide is a promising method for sensitive and accurate identification of HER2+ primary and metastatic lesions. 99mTc‐anti‐HER2 scFv can specifically target malignancies and be used for diagnosis of the cancer type and metastasis as well as treatment of breast cancer. We radiolabeled anti‐HER2 scFv that was expressed in Escherichia coli and purified through Ni‐NTA resin under native condition with 99mTc‐tricarbonyl formed from boranocarbonate. HER2‐based ELISA, BCA, TLC, and HPLC were used in this study. In the current study, anti‐HER2 scFv was lyophilized before radiolabeling. It was found that freeze‐drying did not change the binding activity of anti‐HER2 scFv to HER2. Results demonstrated direct anti‐HER2 scFv radiolabeling by 99mTc‐tricarbonyl to hexahistidine sequence (His‐tag) without any changes in biological activity and radiochemical purity of around 98%. Stability analysis revealed that 99mTc‐anti‐HER2 scFv is stable for at least 24 h in PBS buffer, normal saline, human plasma proteins, and histidine solution.

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“…Reducing the IPTG concentration below 0.1 mM can be employed to increase the delay during induction. In some cases, optimizing the IPTG concentration during induction has little effect on protein expression levels [18,19,[25][26][27]. Systems using lac-based promoters are the most potent and well-studied expression systems.…”

Section: Inducermentioning

confidence: 99%

Additivities for Soluble Recombinant Protein Expression in Cytoplasm of Escherichia coli

Atroshenko,

Sergeev,

Golovina

et al. 2024

Fermentation

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Recombinant protein expression in Escherichia coli is a fundamental technique in molecular biology and biotechnology. This review provides a comprehensive overview of various additivities to enhance the expression levels of soluble recombinant proteins in E. coli. The discussion encompasses five key aspects. Inducer Optimization: strategies for optimizing the inducer concentration to enhance protein expression. Autoinduction system optimization: the examination of glucose, lactose, and glycerol optimization within autoinduction systems to improve protein production. Osmolytes and osmoprotectants: an analysis of the use of osmolytes and osmoprotectants, such as sorbitol and glycine-betaine, to overcome with ease osmotic stress and enhance protein solubility. Ethanol additives: the impact of ethanol on E. coli physiology and its potential to improve recombinant protein expression. Cofactors and metabolic precursors: insights into the addition of cofactors, such as pyridoxal phosphate, riboflavin, thiamine, and pyridoxine, and the utilization of metabolic precursors to enhance the corresponding protein expression. This review highlights both the successful strategies and challenges in recombinant protein expression and provides insights into potential future research directions. Understanding and optimizing these factors is crucial for the efficient production of recombinant proteins for various applications in biotechnology. Furthermore, based on the analyzed data, we propose a straightforward scheme to optimize the additives in the cultivation medium.

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The improvement of anti-HER2 scFv soluble expression in Escherichia coli

Cited by 10 publications

References 26 publications

Noncanonical amino acids as doubly bio-orthogonal handles for one-pot preparation of protein multiconjugates

Noncanonical amino acids as doubly bio-orthogonal handles for one-pot preparation of protein multiconjugates

Preparation, Characterization, and Radiolabeling of Anti‐HER2 scFv With Technetium Tricarbonyl and Stability Studies

Additivities for Soluble Recombinant Protein Expression in Cytoplasm of Escherichia coli

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