Cytoplasmic production of soluble and functional single-chain Fv-Fc fusion protein in Escherichia coli

Sonoda, Hidenori; Kumada, Yoichi; Katsuda, Tomohisa; Yamaji, Hideki

doi:10.1016/j.bej.2010.11.003

Cited by 6 publications

(2 citation statements)

References 32 publications

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“…The formation of disulfide bonds in E. coli can be catalysed in either the naturally oxidative periplasmic compartment 12 or in the cytoplasm of genetically engineered strains 13 14 . Indeed, many fragments derived from mAbs such as Fab 15 , single-chain Fv (scFv) 16 , Fc 17 and an scFv–Fv fusion 18 have been expressed in the periplasm or in the cytoplasm of specially engineered E. coli 19 20 21 . There have even been two reports describing expression and functional assembly of full-length IgGs in E. coli , both in the periplasm 22 23 .…”

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confidence: 99%

Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria

et al. 2015

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Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs—named ‘cyclonals'—effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation.

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Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria

et al. 2015

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“…In particular, non-native aggregation of proteins presents a significant challenge at all stages of biologics production [12–16]. Although strategies were developed to modulate protein aggregation in vitro in bacterial cells [17–20], it is not straightforward to apply them to mammalian cells due to differences in protein synthesis and folding machinery [21]. Therefore, the screening of protein variants in mammalian cells is beneficial to identify protein variants with a reduced aggregation propensity in mammalian cells.…”

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confidence: 99%

Suppressing mutation-induced protein aggregation in mammalian cells by mutating residues significantly displaced upon the original mutation

Gregoire¹,

Glitzos²,

Kwon³

2014

Biochemical Engineering Journal

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Mutations introduced to wild-type proteins naturally, or intentionally via protein engineering, often lead to protein aggregation. In particular, protein aggregation within mammalian cells has significant implications in the disease pathology and biologics production; making protein aggregation modulation within mammalian cells a very important engineering topic. Previously, we showed that the semi-rational design approach can be used to reduce the intracellular aggregation of a protein by recovering the conformational stability that was lowered by the mutation. However, this approach has limited utility when no rational design approach to enhance conformational stability is readily available. In order to overcome this limitation, we investigated whether the modification of residues significantly displaced upon the original mutation is an effective way to reduce protein aggregation in mammalian cells. As a model system, human copper, zinc superoxide dismutase mutant containing glycine to alanine mutation at position 93 (SOD1G93A) was used. A panel of mutations was introduced into residues substantially displaced upon the G93A mutation. By using cell-based aggregation assays, we identified several novel variants of SOD1G93A with reduced aggregation propensity within mammalian cells. Our findings successfully demonstrate that the aggregation of a mutant protein can be suppressed by mutating the residues significantly displaced upon the original mutation.

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Soluble Expression and Characterization of a New scFv Directed to Human CD123

Moradi‐Kalbolandi

Davani

Golkar

et al. 2016

Appl Biochem Biotechnol

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Leukemic cancer stem cells (LSCs), as a unique cell population in acute myeloid leukemia (AML) marked by CD123 overexpression, are thought to play a key role in relapsed AML after chemotherapy. Thus, CD123 is considered as a particularly important target candidate for antibody-derived diagnosis and therapy. In the present work, we constructed an immunized murine antibody phage display library and isolated the functional anti-CD123 Single-chain fragment variable (scFv) clones. We also introduced fusing variable light (VL) and heavy (VH) chains with a new 18-amino acid residue linker as an alternative to conventional linkers. CD123-specific phage clones were progressively enriched through 4 rounds of biopanning, validated by phage ELISA, and anti-CD123 scFv clones with highest affinity were produced in Escherichia coli. The expression and purification of soluble scFv were verified by Western blot, and the results were indicative of the functionality of our proposed linker. The purified scFv specifically recognized CD123 by ELISA and flow cytometry, without any cross-reactivity with other related cell markers. Affinity of anti-CD123 scFv was measured to be 6.9 × 10(-7) M, using the competitive ELISA. Our work, therefore, provides a framework for future studies involving biological functions and applications of our anti-CD123 scFv. It also reveals the feasibility of high throughput methods to isolate biomarker-specific scFvs.

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Cytoplasmic production of soluble and functional single-chain Fv-Fc fusion protein in Escherichia coli

Cited by 6 publications

References 32 publications

Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria

Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria

Suppressing mutation-induced protein aggregation in mammalian cells by mutating residues significantly displaced upon the original mutation

Soluble Expression and Characterization of a New scFv Directed to Human CD123

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