Production of antibody-fragments with plasmid-based and genome-integrated T7 E. coli expression systems – evaluation of systems performance in microtiter fed-batch-like cultivations

Fink, Mathias; Vazulka, Sophie; Jarmer, Johanna; Cserjan‐Puschmann, Monika; Striedner, Gerald

doi:10.1016/j.nbt.2018.05.139

Cited by 3 publications

(4 citation statements)

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“…Cell Disintegration. Cell disintegration and protein extraction were performed according to Fink et al 50 For extraction of all recombinant proteins except for Fragment antigen binding proteins, NuPAGE Sample Reducing Agent (10×) (Novex) was additionally added to a concentration of 4 mM to the lysis buffer, respectively.…”

Section: ■ Methodsmentioning

confidence: 99%

“…Quantification of Fragment Antigen Binding Protein. Soluble fractions of Fab against VEGF-A were quantified according to Fink et al 50 Quantification of HIV-1pr, hPTH, Protein P1/P2. Soluble and insoluble recombinant protein fractions were determined by densitometry analysis of SDS-PAGE gel using known concentrations of bovine serum albumin (BSA) for establishing a calibration curve.…”

Section: ■ Methodsmentioning

confidence: 99%

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Bacteriophage Inspired Growth-Decoupled Recombinant Protein Production in Escherichia coli

Stargardt¹,

Feuchtenhofer²,

Cserjan‐Puschmann

et al. 2020

ACS Synth. Biol.

100

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Modulating resource allocation in bacteria to redirect metabolic building blocks to the formation of recombinant proteins rather than biomass formation remains a grand challenge in biotechnology. Here, we present a novel approach for improved recombinant protein production (RPP) using Escherichia coli (E. coli) by decoupling recombinant protein synthesis from cell growth. We show that cell division and host mRNA transcription can be successfully inhibited by coexpression of a bacteriophagederived E. coli RNA polymerase (RNAP) inhibitor peptide and that genes overtranscribed by the orthogonal T7 RNAP can finally account to >55% of cell dry mass (CDM). This RNAP inhibitor peptide binds the E. coli RNAP and therefore prevents σ-factor 70 mediated formation of transcriptional qualified open promoter complexes. Thereby, the transcription of σ-factor 70 driven host genes is inhibited, and metabolic resources can be exclusively utilized for synthesis of the protein of interest (POI). Here, we mimic the late phase of bacteriophage infection by coexpressing a phage-derived xenogeneic regulator that reprograms the host cell and thereby are able to significantly improve RPP under industrial relevant fed-batch process conditions at bioreactor scale. We have evaluated production of several different recombinant proteins at different scales (from microscale to 20 L fed-batch scale) and have been able to improve total and soluble proteins yields up to 3.4fold in comparison to the reference expression system E. coli BL21(DE3). This novel approach for growth-decoupled RPP has profound implications for biotechnology and bioengineering and helps to establish more cost-effective and generic manufacturing processes for biologics and biomaterials.

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Section: ■ Methodsmentioning

confidence: 99%

Section: ■ Methodsmentioning

confidence: 99%

Bacteriophage Inspired Growth-Decoupled Recombinant Protein Production in Escherichia coli

Stargardt¹,

Feuchtenhofer²,

Cserjan‐Puschmann

et al. 2020

ACS Synth. Biol.

100

View full text Add to dashboard Cite

show abstract

“…For intracellular product analysis, cell disintegration, fractionation of soluble and insoluble protein, and IB solubilization were performed according to Fink et al . 20 . NuPAGE Sample Reducing Agent (10×) (Invitrogen, Waltham, MA, USA) was added additionally to a concentration of 4 mmol L −1 to the lysis buffer.…”

Section: Methodsmentioning

confidence: 99%

“…For intracellular product analysis, cell disintegration, fractionation of soluble and insoluble protein, and IB solubilization were performed according to Fink et al. 20 NuPAGE Sample Reducing Agent (10×) (Invitrogen, Waltham, MA, USA) was added additionally to a concentration of 4 mmol L −1 to the lysis buffer. For semi-quantitative determination of FGF2, 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to Stargardt et al 21 and Cserjan-Puschmann et al 14 was employed.…”

Section: Fermentationmentioning

confidence: 99%

Advanced purification platform using circularly permuted caspase‐2 for affinity fusion‐tag removal to produce native fibroblast growth factor 2

Lingg

Cserjan‐Puschmann

Fischer

et al. 2021

J of Chemical Tech & Biotech

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BACKGROUND: Recombinant proteins produced for use as biopharmaceuticals need to harbor their native N-terminus. A drawback in expression of recombinant proteins as fusion proteins with an affinity fusion-tag is that enzymatic or chemical processing is required to trim the artificial tag and release the true protein of interest. In many cases, however, this processing step generates an incorrect N-terminus.RESULTS: Human fibroblast growth factor 2 (FGF2) was expressed as a fusion protein in Escherichia coli fed-batch cultivations. The protein of interest (POI) carried an N-terminal affinity fusion-tag which enabled purification via affinity chromatography. After enzymatic removal of the affinity fusion-tag with a circularly permuted human caspase-2 (cpCasp2), the POI was further purified using subtractive affinity chromatography. Mass spectrometric analysis confirmed the authentic N-terminus of the POI. The generated POI was highly pure with 42 ppm host cell protein, 3.7 ∼g mL −1 dsDNA and ∼ 1000 EU mL −1 endotoxin. Only a small number of E. coli host cell proteins were co-purified with the POI. Because of the high specificity of the novel protease cpCasp2, no off-target cleavage could be observed. CONCLUSION: Our findings demonstrate that cpCasp2 can be used for the production of native proteins using a fusionprotein process. This represents a first case study at large laboratory scale for the production of an industrially relevant protein. This technology constitutes the basis of a highly scalable cpCasp2-based platform fusion protein process (CASPON technology) purification platform.

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Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization

et al. 2020

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Caspase-2 is the most specific protease of all caspases and therefore highly suitable as tag removal enzyme creating an authentic N-terminus of overexpressed tagged proteins of interest. The wild type human caspase-2 is a dimer of heterodimers generated by autocatalytic processing which is required for its enzymatic activity. We designed a circularly permuted caspase-2 (cpCasp2) to overcome the drawback of complex recombinant expression, purification and activation, cpCasp2 was constitutively active and expressed as a single chain protein. A 22 amino acid solubility tag and an optimized fermentation strategy realized with a model-based control algorithm further improved expression in Escherichia coli and 5.3 g/L of cpCasp2 in soluble form were obtained. The generated protease cleaved peptide and protein substrates, regardless of N-terminal amino acid with high activity and specificity. Edman degradation confirmed the correct N-terminal amino acid after tag removal, using Ubiquitin-conjugating enzyme E2 L3 as model substrate. Moreover, the generated enzyme is highly stable at −20 °C for one year and can undergo 25 freeze/thaw cycles without loss of enzyme activity. The generated cpCasp2 possesses all biophysical and biochemical properties required for efficient and economic tag removal and is ready for a platform fusion protein process.

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Production of antibody-fragments with plasmid-based and genome-integrated T7 E. coli expression systems – evaluation of systems performance in microtiter fed-batch-like cultivations

Cited by 3 publications

References 0 publications

Bacteriophage Inspired Growth-Decoupled Recombinant Protein Production in Escherichia coli

Bacteriophage Inspired Growth-Decoupled Recombinant Protein Production in Escherichia coli

Advanced purification platform using circularly permuted caspase‐2 for affinity fusion‐tag removal to produce native fibroblast growth factor 2

Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization

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