Purification optimization for a recombinant single-chain variable fragment against type 1 insulin-like growth factor receptor (IGF-1R) by using design of experiment (DoE)

Song, Yonghong; Sun, Xuewen; Jiang, Bo; Liu, Ji-En; Su, Xianhui

doi:10.1016/j.pep.2015.08.020

Cited by 9 publications

(2 citation statements)

References 14 publications

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“…60 When expressed recombinantly, cell-wall anchoring domains of the wild type protein are typically omitted, and some truncated versions have only 3 or 4 light chain binding domains. Protein L is routinely used for the purification of scFv constructs 61 and has been employed as a universal flow cytometry marker for cells expressing chimeric antigen receptors (CARs), 62 which typically utilize scFv segments to recognize their targets. Because Protein L binds to variable light chains without interfering with the antigen recognition loops, it can be used as a "secondary" detection reagent for bound scFv's and IgG's.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins

et al. 2020

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Oxidative coupling (OC) through o-quinone intermediates has been established as an efficient and site-selective way to modify protein N-termini and the unnatural amino acid p-aminophenylalanine (paF). Recently, we reported that the tyrosinase-mediated oxidation of phenol-tagged cargo molecules is a particularly convenient method of generating o-quinones in situ. The coupling partners can be easily prepared and stored, the reaction takes place under mild conditions (phosphate buffer, pH 6.5, 4 to 23 °C), and dissolved oxygen is the only oxidant required. Here, we show an important extension of this chemistry for the activation of tyrosine residues that project into solution from the N or C-termini of peptide and protein substrates. Generating the o-quinone electrophiles from tyrosine allows greater flexibility in choosing the nucleophilic coupling partner and expands the scope of the reaction to include C-terminal positions. We also introduce a new bacterial tyrosinase enzyme that shows improved activation for some tyrosine substrates. The efficacy of several secondary amines and aniline derivatives was evaluated in the coupling reactions, providing important information for coupling partner design. This strategy was used to modify the C-termini of an antibody scFv construct and of Protein L, a human IgG kappa light chain binding protein. The use of the modified proteins as immunolabeling agents was also demonstrated.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins

et al. 2020

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“…When expressed recombinantly, cell-wall anchoring domains of the wild type protein are typically omitted, and some truncated versions have only 3 or 4 light chain binding domains. Protein L is routinely used for the purification of scFv constructs 57 and has been employed as a universal flow cytometry marker for cells expressing Chimeric Antigen Receptors (CARs) 58 , which typically utilize scFv segments to recognize their targets. Because Protein L binds to variable light chains without interfering with the antigen recognition loops, it can be used as a "secondary" detection reagent for bound scFv's and IgG's.…”

Section: Resultsmentioning

confidence: 99%

Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins

Marmelstein¹,

Lobba²,

Mogilevsky³

et al. 2019

Preprint

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We report a strategy for chemical protein modification by using tyrosinase enzymes to oxidize exposed tyrosine residues on protein N or C-termini. We explore the chemical space for coupling partners in this reaction and find combinations that can proceed in near quantitative conversion. This strategy is used to conjugate a dye onto a Trastuzumab antibody fragment and a Protein L fragment and demonstrate that these constructs can be used as immunostaining reagents.

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Clear insight into complex multimodal resins and impurities to overcome recombinant protein purification challenges: A review

Goodarzi,

Jalalirad

2024

Biotech & Bioengineering

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Increasing attention has been paid to the purity of therapeutic proteins imposing extensive costs and challenges to the downstream processing of biopharmaceuticals. One of the efforts, that has been exerted to overcome such limitations, was developing multimodal or mixed‐mode chromatography (MMC) resins for launching selective, orthogonal, non‐affinity purification platforms. Despite relatively extensive usage of MMC resins, their real potential and fulfillment have not been extensively reviewed yet. In this work, the explanation of practical and key aspects of downstream processing of recombinant proteins with or without MMC resins was debated, as being useful for further purification process development. This review has been written as a step‐by‐step guide to deconvolute both inherent protein purification and MMC complexities. Here, after complete elucidation of the potential of MMC resins, the effects of frequently used additives (mobile phase modifiers) and their possible interactions during the purification process, the critical characteristics of common product‐related impurities (e.g., aggregates, charge variants, fragments), host‐related impurities (e.g., host cell protein and DNA) and process related impurities (e.g., endotoxin, and viruses) with solved or unsolved challenges of traditional and MMC resins have been discussed. Such collective experiences which are reported in this study could be considered as an applied guide for developing successful downstream processing in challenging conditions by providing a clear insight into complex MMC resins and impurities.

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Purification optimization for a recombinant single-chain variable fragment against type 1 insulin-like growth factor receptor (IGF-1R) by using design of experiment (DoE)

Cited by 9 publications

References 14 publications

Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins

Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins

Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins

Clear insight into complex multimodal resins and impurities to overcome recombinant protein purification challenges: A review

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