Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins

AbstractIn crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over PCR-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a dsDNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based Protection of LET) method effectively increases sfGFP expression levels from LETs in Escherichia coli CFPS reactions by 6-fold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from non-model organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.

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Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.64.12.4862-4869.1998 ◽

1998 ◽

Vol 64 (12) ◽

pp. 4862-4869 ◽

Cited By ~ 39

Author(s):

Jörg F. Rippmann ◽

Michaela Klein ◽

Christian Hoischen ◽

Bodo Brocks ◽

Wolfgang J. Rettig ◽

...

Keyword(s):

Escherichia Coli ◽

Proteus Mirabilis ◽

Binding Activity ◽

Host Cells ◽

Heterologous Proteins ◽

Single Chain Variable Fragment ◽

Single Chain ◽

E Coli ◽

Active Product ◽

Periplasmic Expression

ABSTRACT Recently it has been demonstrated that L-form cells ofProteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coliJM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

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Solubility assessment of single-chain antibody fragment against epithelial cell adhesion molecule extracellular domain in four Escherichia coli strains

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00126-1 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Fatemeh Sadat Javadian ◽

Majid Basafa ◽

Aidin Behravan ◽

Atieh Hashemi

Keyword(s):

Escherichia Coli ◽

Cell Adhesion ◽

Epithelial Cell ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Extracellular Domain ◽

Epithelial Cell Adhesion Molecule ◽

Single Chain ◽

E Coli ◽

The Impact

Abstract Background Overexpression of the EpCAM (epithelial cell adhesion molecule) in malignancies makes it an attractive target for passive immunotherapy in a wide range of carcinomas. In comparison with full-length antibodies, due to the small size, the scFvs (single-chain variable fragments) are more suitable for recombinant expression in E. coli (Escherichia coli). However, the proteins expressed in large amounts in E. coli tend to form inclusion bodies that need to be refolded which may result in poor recovery of bioactive proteins. Various engineered strains were shown to be able to alleviate the insolubility problem. Here, we studied the impact of four E. coli strains on the soluble level of anti-EpEX-scFv (anti-EpCAM extracellular domain-scFv) protein. Results Although results showed that the amount of soluble anti-EpEX-scFv obtained in BL21TM (DE3) (114.22 ± 3.47 mg/L) was significantly higher to those produced in the same condition in E. coli RosettaTM (DE3) (71.39 ± 0.31 mg/L), and OrigamiTM T7 (58.99 ± 0.44 mg/L) strains, it was not significantly different from that produced by E. coli SHuffleTM T7 (108.87 ± 2.71 mg/L). Furthermore, the highest volumetric productivity of protein reached 318.29 ± 26.38 mg/L in BL21TM (DE3). Conclusions Although BL21TM (DE3) can be a suitable strain for high-level production of anti-EpEX-scFv protein, due to higher solubility yield (about 55%), E. coli SHuffleTM T7 seems to be better candidate for soluble production of scfv compared to BL21TM (DE3) (solubility yield of about 30%).

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Detection of Escherichia coli O157, Salmonella enterica Serovar Typhimurium, and Staphylococcal Enterotoxin B in a Single Sample Using Enzymatic Bio-Nanotransduction

Journal of Food Protection ◽

10.4315/0362-028x-70.4.841 ◽

2007 ◽

Vol 70 (4) ◽

pp. 841-850 ◽

Cited By ~ 8

Author(s):

JOSH R. BRANEN ◽

MARTHA J. HASS ◽

ERIN R. DOUTHIT ◽

WUSI C. MAKI ◽

A. LARRY BRANEN

Keyword(s):

Escherichia Coli ◽

Salmonella Enterica ◽

Salmonella Enterica Serovar Typhimurium ◽

Staphylococcal Enterotoxin B ◽

Escherichia Coli O157 ◽

Dna Templates ◽

E Coli ◽

Serovar Typhimurium ◽

Biological Recognition ◽

Enterotoxin B

Enzymatic bio-nanotransduction is a biological detection scheme based on the production of nucleic acid nano-signals (RNA) in response to specific biological recognition events. In this study, we applied an enzymatic bio-nanotransduction system to the detection of important food-related pathogens and a toxin. Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and staphylococcal enterotoxin B (SEB) were chosen because of the implications of these targets to food safety. Primary antibodies to each of the targets were used to functionalize magnetic beads and produce biological recognition elements (antibodies) conjugated to nano-signal–producing DNA templates. Immunomagnetic capture that was followed by in vitro transcription of DNA templates bound to target molecules produced RNA nano-signals specific for every target in the sample. Discrimination of RNA nano-signals with a standard enzyme-linked oligonucleotide fluorescence assay provided a correlation between nano-signal profiles and target concentrations. The estimated limit of detection was 2.4 × 103 CFU/ml for E. coli O157:H7, 1.9 × 104 CFU/ml for S. enterica serovar Typhimurium, and 0.11 ng/ml for SEB with multianalyte detection in buffer. Low levels of one target were also detected in the presence of interference from high levels of the other targets. Finally, targets were detected in milk, and detection was improved for E. coli O157 by heat treatment of the milk.

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Expression in Escherichia Coli of a Single - Chain Variable - Fragment ( scFv ) Against the Amino Acid Motif DELLA of Plant Transcription Factor Is Toxic to E. Coli

International Journal for Sciences and Technology ◽

10.12816/0010080 ◽

2013 ◽

Vol 8 (4) ◽

pp. 19-26

Author(s):

Taha Al-Samarrai

Keyword(s):

Escherichia Coli ◽

Transcription Factor ◽

Amino Acid ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Amino Acid Motif ◽

E Coli ◽

Expression In Escherichia Coli

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Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5′-Terminal AUG

Journal of Bacteriology ◽

10.1128/jb.00091-17 ◽

2017 ◽

Vol 199 (14) ◽

Cited By ~ 5

Author(s):

Heather J. Beck ◽

Gary R. Janssen

Keyword(s):

Escherichia Coli ◽

Translation Initiation ◽

Translational Regulation ◽

Current Knowledge ◽

Upstream Open Reading Frame ◽

Model Organisms ◽

Regulation Mechanism ◽

Rna Regulation ◽

Content Type ◽

E Coli

ABSTRACT Alternative translation initiation mechanisms, distinct from the Shine-Dalgarno (SD) sequence-dependent mechanism, are more prevalent in bacteria than once anticipated. Translation of Escherichia coli ptrB instead requires an AUG triplet at the 5′ terminus of its mRNA. The 5′-terminal AUG (5′-uAUG) acts as a ribosomal recognition signal to attract ribosomes to the ptrB mRNA rather than functioning as an initiation codon to support translation of an upstream open reading frame. ptrB expression exhibits a stronger dependence on the 5′-uAUG than the predicted SD sequence; however, strengthening the predicted ptrB SD sequence relieves the necessity for the 5′-uAUG. Additional sequences within the ptrB 5′ untranslated region (5′-UTR) work cumulatively with the 5′-uAUG to control expression of the downstream ptrB coding sequence (CDS), thereby compensating for the weak SD sequence. Replacement of 5′-UTRs from other mRNAs with the ptrB 5′-UTR sequence showed a similar dependence on the 5′-uAUG for CDS expression, suggesting that the regulatory features contained within the ptrB 5′-UTR are sufficient to control the expression of other E. coli CDSs. Demonstration that the 5′-uAUG present on the ptrB leader mRNA is involved in ribosome binding and expression of the downstream ptrB CDS revealed a novel form of translational regulation. Due to the abundance of AUG triplets at the 5′ termini of E. coli mRNAs and the ability of ptrB 5′-UTR regulation to function independently of gene context, the regulatory effects of 5′-uAUGs on downstream CDSs may be widespread throughout the E. coli genome. IMPORTANCE As the field of synthetic biology continues to grow, a complete understanding of basic biological principles will be necessary. The increasing complexity of the synthetic systems highlights the gaps in our current knowledge of RNA regulation. This study demonstrates that there are novel ways to regulate canonical Shine-Dalgarno-led mRNAs in Escherichia coli, illustrating that our understanding of the fundamental processes of translation and RNA regulation is still incomplete. Even for E. coli, one of the most-studied model organisms, genes with translation initiation mechanisms that do not fit the canonical Shine-Dalgarno sequence paradigm are being revealed. Uncovering diverse mechanisms that control translational expression will allow synthetic biologists to finely tune protein production of desired gene products.

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Specific Secretion of Active Single-Chain Fv Antibodies into the Supernatants of Escherichia coliCultures by Use of the Hemolysin System

Applied and Environmental Microbiology ◽

10.1128/aem.66.11.5024-5029.2000 ◽

2000 ◽

Vol 66 (11) ◽

pp. 5024-5029 ◽

Cited By ~ 55

Author(s):

Luis A. Fernández ◽

Isabel Sola ◽

Luis Enjuanes ◽

Víctor de Lorenzo

Keyword(s):

Escherichia Coli ◽

Transport System ◽

Culture Media ◽

Single Chain ◽

Extracellular Medium ◽

Simple Method ◽

E Coli ◽

Single Chain Fv ◽

Bacterial Cytoplasm ◽

Culture Supernatants

ABSTRACT A simple method for the nontoxic, specific, and efficient secretion of active single-chain Fv antibodies (scFvs) into the supernatants ofEscherichia coli cultures is reported. The method is based on the well-characterized hemolysin transport system (Hly) of E. coli that specifically secretes the target protein from the bacterial cytoplasm into the extracellular medium without a periplasmic intermediate. The culture media that accumulate these Hly-secreted scFv's can be used in a variety of immunoassays without purification. In addition, these culture supernatants are stable over long periods of time and can be handled basically as immune sera.

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Neutralizationof Enteric Coronaviruses with Escherichia coli CellsExpressing Single-Chain Fv-AutotransporterFusions

Journal of Virology ◽

10.1128/jvi.77.24.13396-13398.2003 ◽

2003 ◽

Vol 77 (24) ◽

pp. 13396-13398 ◽

Cited By ~ 14

Author(s):

Esteban Veiga ◽

Víctor de Lorenzo ◽

Luis Angel Fernández

Keyword(s):

Escherichia Coli ◽

Infectious Agent ◽

Target Cells ◽

Single Chain ◽

E Coli ◽

Single Chain Antibodies ◽

Single Chain Fv ◽

Transmissible Gastroenteritis ◽

Iga Protease

ABSTRACT We report here that fusions of single-chain antibodies (scFvs) to the autotransporter β domain of the IgA protease of Neisseria gonorrhoeae are instrumental in locating virus-neutralizing activity on the cell surface of Escherichia coli. E. coli cells displaying scFvs against the transmissible gastroenteritis coronavirus on their surface blocked in vivo the access of the infectious agent to cultured epithelial cells. This result raises prospects for antiviral strategies aimed at hindering the entry into target cells by bacteria that naturally colonize the same intestinal niches.

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Inexpensive and colorimetric RNA detection by E. coli cell-free protein synthesis platform at room temperature

10.1101/2021.11.29.21267025 ◽

2021 ◽

Author(s):

Michela Notarangelo ◽

Alessandro Quattrone ◽

Massimo Pizzato ◽

Sheref S. Mansy ◽

O. Duhan Toparlak

Keyword(s):

Room Temperature ◽

Colorimetric Detection ◽

In Vitro Transcription ◽

Viral Rna ◽

Rna Sequences ◽

Rna Detection ◽

E Coli ◽

Free Gene ◽

Cell Free Protein Synthesis

We report colorimetric detection of SARS-CoV-2 viral RNA by an in vitro transcription/translation assay with crude E. coli extracts at room temperature, with the aid of body heat. Clinically-relevant concentrations of viral RNA (ca. 600 copies/test) were detected from synthetic RNA samples. The activation of cell-free gene expression was achieved by toehold-switch-mediated riboregulatory elements that are specific to viral RNA sequences. The colorimetric output was generated by the α-complementation of β-galactosidase ω-fragment (LacZ-ω) with cell-free expressed LacZ-α, using an X-gal analogue as a substrate. The estimated cost of single reaction is less than 1 euro/test, which may facilitate diagnostic kit accessibility in developing countries.

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Rapid production and characterization of antimicrobial colicins using Escherichia coli-based cell-free protein synthesis

Synthetic Biology ◽

10.1093/synbio/ysy004 ◽

2018 ◽

Vol 3 (1) ◽

Cited By ~ 12

Author(s):

Xing Jin ◽

Weston Kightlinger ◽

Yong-Chan Kwon ◽

Seok Hoon Hong

Keyword(s):

Escherichia Coli ◽

Protein Synthesis ◽

Host Cells ◽

Persister Cells ◽

Free Protein ◽

E Coli ◽

Dna And Rna ◽

Rapid Production ◽

Cell Free Protein Synthesis

Abstract Colicins are antimicrobial proteins produced by Escherichia coli, which, upon secretion from the host, kill non-host E. coli strains by forming pores in the inner membrane and degrading internal cellular components such as DNA and RNA. Due to their unique cell-killing activities, colicins are considered viable alternatives to conventional antibiotics. Recombinant production of colicins requires co-production of immunity proteins to protect host cells; otherwise, the colicins are lethal to the host. In this study, we used cell-free protein synthesis (CFPS) to produce active colicins without the need for protein purification and co-production of immunity proteins. Cell-free synthesized colicins were active in killing model E. coli cells with different modes of cytotoxicity. Pore-forming colicins E1 and nuclease colicin E2 killed actively growing cells in a nutrient-rich medium, but the cytotoxicity of colicin Ia was low compared to E1 and E2. Moreover, colicin E1 effectively killed cells in a nutrient-free solution, while the activity of E2 was decreased compared to nutrient-rich conditions. Both colicins E1 and E2 decreased the level of persister cells (metabolically dormant cell populations that are insensitive to antibiotics) by up to six orders of magnitude compared to that of the rifampin pretreated persister cells. This study finds that colicins can eradicate non-growing cells including persisters, and that CFPS is a promising platform for rapid production and characterization of toxic proteins.

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