Production of antibodies in SHuffle Escherichia coli strains

Eaglesham, J.B.; García, Augusto F.; Berkmen, Mehmet

doi:10.1016/bs.mie.2021.06.040

Cited by 9 publications

(5 citation statements)

References 127 publications

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“…Successful cytoplasmic expression of IgGs in E. coli has previously been described, but titers are typically very low (<20 mg/L in shake flasks). 13 , 16 , 25 , 32 To produce IgGs in SBDG419, a pJ411-based expression plasmid was designed that contained the HC and LC sequences of an anti-CD74 IgG in a bicistronic operon, with the HC preceding the LC. To ensure sufficient expression of the LC, an additional ribosome-binding site 5’ to the LC start codon was included.…”

Section: Resultsmentioning

confidence: 99%

“…While others have reported the successful production of antibodies and antibody fragments in the E. coli cytoplasm, the reported titers are typically low (<200 mg/L). 13 , 16 Strain SBDG419 is similar to strains used in previous reports in which the cytoplasmic glutathione reductase (gor) and thiodoxin reductase (trxB) are knocked out and the disulfide bond isomerase, dsbC, is expressed in the cytoplasm. 37 However, SBDG419 was constructed with an additional knock out to the thioredoxin trxA, which was previously shown to result in higher yields of cytoplasmically-expressed proteins that contain disulfide bonds.…”

Section: Discussionmentioning

confidence: 99%

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Production of antibodies and antibody fragments containing non-natural amino acids in Escherichia coli

Blake-Hedges,

Groff,

Foo

et al. 2024

mAbs

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Therapeutic bioconjugates are emerging as an essential tool to combat human disease. Site-specific conjugation technologies are widely recognized as the optimal approach for producing homogeneous drug products. Non-natural amino acid (nnAA) incorporation allows the introduction of bioconjugation handles at genetically defined locations. Escherichia coli ( E. coli ) is a facile host for therapeutic nnAA protein synthesis because it can stably replicate plasmids encoding genes for product and nnAA incorporation. Here, we demonstrate that by engineering E. coli to incorporate high levels of nnAAs, it is feasible to produce nnAA-containing antibody fragments and full-length immunoglobulin Gs (IgGs) in the cytoplasm of E. coli . Using high-density fermentation, it was possible to produce both of these types of molecules with site-specifically incorporated nnAAs at titers > 1 g/L. We anticipate this strategy will help simplify the production and manufacture of promising antibody therapeutics.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Production of antibodies and antibody fragments containing non-natural amino acids in Escherichia coli

Blake-Hedges,

Groff,

Foo

et al. 2024

mAbs

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“…More recently, antibodies have been produced in semi-oxidized SHuffle cytoplasm as soluble proteins without the need for renaturation and refolding. 16 Recent progress in proteomics and metabolomics combined with systems and synthetic biology approaches could be harnessed in E. coli to improve the titer and quality of antibodies and to reduce manufacturing costs compared with that achievable with mammalian cell-based processes. The E. coli -produced aglycosylated antibodies should, in fact, be preferable over their CHO-produced counterparts considering their homogeneity (due to the lack of glycan isoforms), cost effectiveness and simplified downstream processing, and suitability for disease indications where effector functions are either unnecessary or unwanted.…”

Section: Discussionmentioning

confidence: 99%

“… 97–100 Many FL-IgGs have been expressed in soluble and functional forms in the cytoplasm of this strain. 16 …”

Section: Production In Escherichia Colimentioning

confidence: 99%

Full-length recombinant antibodies from Escherichia coli : production, characterization, effector function (Fc) engineering, and clinical evaluation

Rashid

2022

mAbs

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Although several antibody fragments and antibody fragment-fusion proteins produced in Escherichia coli ( E. coli ) are approved as therapeutics for various human diseases, a full-length monoclonal or a bispecific antibody produced in E. coli has not yet been approved. The past decade witnessed substantial progress in expression of full-length antibodies in the E. coli cytoplasm and periplasm, as well as in cell-free expression systems. The equivalency of E. coli -produced aglycosylated antibodies and their mammalian cell-produced counterparts, with respect to biochemical and biophysical properties, including antigen binding, in vitro and in vivo serum stability, pharmacokinetics, and in vivo serum half-life, has been demonstrated. Extensive engineering of the Fc domain of aglycosylated antibodies enables recruitment of various effector functions, despite the lack of N -linked glycans. This review summarizes recent research, preclinical advancements, and clinical development of E. coli -produced aglycosylated therapeutic antibodies as monoclonal, bispecific, and antibody-drug conjugates for use in autoimmune, oncology, and immuno-oncology areas. Abbreviations: ADA Anti-drug antibody; ADCC Antibody-dependent cellular cytotoxicity; ADCP Antibody-dependent cellular phagocytosis; ADC Antibody-drug conjugate; aFc Aglycosylated Fc; AMD Age-related macular degeneration aTTP Acquired thrombotic thrombocytopenic purpura; BCMA B-cell maturation antigen; BLA Biologics license application; BsAb Bispecific antibody; C1q Complement protein C1q; CDC Complement-dependent cytotoxicity; CDCC Complement-dependent cellular cytotoxicity; CDCP Complement-dependent cellular phagocytosis; CEX Cation exchange chromatography; CFPS Cell-free protein expression; CHO Chinese Hamster Ovary; CH1-3 Constant heavy chain 1-3; CL Constant light chain; DLBCL Diffuse large B-cell lymphoma; DAR Drug antibody ratio; DC Dendritic cell; dsFv Disulfide-stabilized Fv; EU European Union; EGFR Epidermal growth factor receptor; E. coli Escherichia coli; EpCAM Epithelial cell adhesion molecule; Fab Fragment antigen binding; FACS Fluorescence activated cell sorting; Fc Fragment crystallizable; FcRn Neonatal Fc receptor; FcɣRs Fc gamma receptors; FDA Food and Drug Administration; FL-IgG Full-length immunoglobulin; Fv Fragment variable; FolRαa Folate receptor alpha; gFc Glycosylated Fc; GM-CSF Granulocyte macrophage-colony stimulating factor; GPx7 Human peroxidase 7; HCL Hairy cell leukemia; HIV Human immunodeficiency virusl; HER2 Human epidermal growth factor receptor 2; HGF Hepatocyte growth factor; HIC Hydrophobic interaction chromatography; HLA Human leukocyte antigen; IBs Inclusion bodies; IgG1-4 Immunoglobulin 1-4; IP Intraperitoneal; ITC Isothermal titration calorimetry; ITP Immune...

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“…SHuffle strains for the recombinant expression of proteins requiring the formation of disulfide bonds in the cytoplasm. E. coli shuffle strains have been engineered to have an oxidizing cytoplasm and to constitutively express a chromosomal copy of the di-sulfide bond isomerase DsbC, promoting the formation of di-sulfide bonds in the cytoplasm 35,36,37 .…”

Section: Case Study 2: Parseq Enables the Retrieval Of Sequence Verif...mentioning

confidence: 99%

parSEQ:Probe and Rescue Sequencing for Advanced Variant Retrieval from DNA Pool

Houmani,

Peterkin,

Antoun

et al. 2023

Preprint

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Modern protein engineering is powered by sequence-function data-sets. We have developed parSEQ, a platform that maximizes the capture of these protein sequence-function data-sets through a sequence-first-screen-later approach. parSEQ relies on Next-Generation Sequencing (NGS) to reverse the conventional screen-first-sequence-later workflow. This allows for the high-throughput retrieval of variant sequences from DNA pools, ensuring that every screened variant’s functional data is paired with its sequence data. This report details parSEQ’s methodology and its integration into various protein engineering workflows. Through several case studies, we illustrate parSEQ’s broad applicability. These case studies describe the use of parSEQ for high-throughput variant DNA-template retrieval, expression-ready bacterial clone isolation, sourcing DNA for denovo designed proteins, and preparing targeted mutational libraries. Our findings suggest that parSEQ’s approach to capturing sequence-function data-sets can advance protein engineering efforts, particularly in the age of artificial intelligence and machine learning.

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Production of antibodies in SHuffle Escherichia coli strains

Cited by 9 publications

References 127 publications

Production of antibodies and antibody fragments containing non-natural amino acids in Escherichia coli

Production of antibodies and antibody fragments containing non-natural amino acids in Escherichia coli

Full-length recombinant antibodies from Escherichia coli : production, characterization, effector function (Fc) engineering, and clinical evaluation

parSEQ:Probe and Rescue Sequencing for Advanced Variant Retrieval from DNA Pool

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