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

1998 ◽  
Vol 64 (12) ◽  
pp. 4862-4869 ◽  
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.

Heparin-Induced Thrombocytopenia and Thrombosis: Therapeutic Strategy Using a Single-Chain Variable Fragment (scFv) Antibody

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3346-3346
Author(s):  
Jaa Yien New ◽  
Jose Perdomo ◽  
Xing-Mai Jiang ◽  
Beng Chong
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Heparin Therapy ◽  
Human Platelets ◽  
Binding Activity ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Heparin Induced Thrombocytopenia ◽  
E Coli

Abstract Abstract 3346 Introduction and Aim Heparin-Induced Thrombocytopenia and Thrombosis (HIT) is a life threatening disorder that affects 1–5% of patients receiving heparin therapy. A low platelet count is usually recorded (<150,000 per cubic millimetre) with a decrease of 50% or more from the baseline. The occurrence of HIT is due to the presence of an IgG antibody that recognizes the immune complex formed between Platelet Factor 4 (PF4) and heparin. The antibody/PF4/Heparin complex binds to the FcγRIIa receptor on platelets, leading to platelet activation and thrombotic complications in patients receiving heparin. IV.3 is a murine monoclonal antibody that was raised against the FcγRIIa receptor and has been used as an inhibitor in specificity assays to confirm HIT in patients. We have developed a humanized single-chain variable fragment (scFv) antibody based on the IV.3 monoclonal antibody that binds to the FcγRIIa receptor on platelets and prevents platelet aggregation induced by HIT antibodies. Methods The variable heavy chain (VH) and light chain (VL) of the IV.3 antigen binding fragment (Fab) moiety were amplified using polymerase chain reaction (PCR). These two fragments were then coupled with a linker (Glycine4 and Serine)6. This was followed by introduction of several components including fusion tags (FLAG and c-Myc) at both termini for cloning, detection and purification purposes. The construct was transformed into E. coli (strain-BL21) for protein expression of the scFv. The presence of the protein was detected via immunostaining using anti-FLAG and anti-c-Myc antibodies. The scFv was purified by affinity chromatography and the binding activity was detected using flow cytometry and confocal microscopy. The functional activity was determined using Platelet Aggregation Assay. The scFv was then humanized to minimize potential immunogenicity. Humanization was achieved by introducing specific mutations that rendered the molecule human-like but did not affect binding specificity. The humanized scFv was also expressed in E. coli, purified and tested as before. Results The scFv protein (32kDa) was expressed, purified and confirmed via immunostaining. The created humanized scFv exhibits binding activity against the FcγRIIa on human platelets as determined by flow cytometry and confocal microscopy. In addition, the protein successfully inhibits platelet aggregation at micro molar concentrations in aggregation assays conducted in vitro in the presence of HIT antibodies. Conclusions The humanized scFv was successful in recapitulating the properties of the IV.3 murine monoclonal antibody. It demonstrated binding activity against the FcgRIIa on human platelets and exhibited functional activity by inhibiting platelet activation and aggregation in vitro. This implies that our scFv is able to stop binding of the antibody/PF4/Heparin immune complex to platelets, thus hindering one of the critical initial steps in HIT. The scFv described here may be able to ameliorate the unwanted side effects of heparin therapy and could serve as a potential therapeutic drug for HIT patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Comparison of Gene Expression Between Two Types of Anti-EGFRvIII ScFv Antibodies Having Different Variable Domain Orders in Escherichia coli

2017 ◽  
Vol 21 (1) ◽  
pp. 29
Author(s):  
Kartika Sari Dewi ◽  
Asrul Muhamad Fuad
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Binding Activity ◽  
Extracellular Proteins ◽  
Antigen Binding ◽  
Dna Encoding ◽  
Single Chain ◽  
Colony Pcr ◽  
E Coli ◽  
Variable Domains

Several studies reported that the expression of various kinds of Single-chain variable fragment (scFv) antibodies in Escherichia coli are significantly influenced by the order of their variable domains. To date, the effect of the order of variable domains in the expression of scFv antibodies against epidermal growth factor receptor variant III (EGFRvIII) has not been reported. This study aimed to compare the expression between VH-linker-VL and VL-linker-VH domain orders of the anti-EGFRvIII scFv antibodies in E. coli expression system. Recombinant plasmids inserted with DNA encoding scFv proteins were transformed into E. coli NiCo21(DE3) competent cells and characterized by colony PCR. The expression of scFv proteins was done by using optimum concentration of inducer. Total proteins, soluble periplasmic and cytoplasmic proteins, also extracellular proteins were isolated, subsequently characterized by SDS-PAGE, Slot Blot, and ImageJ software analyses. The antigen-binding activity of both scFvs proteins against EGFRvIII was observed. The results showed that the relative percentage of scFv expression with VH-linker-VL domain order is higher than that of VL-linker-VH in each compartment. Moreover, both of scFvs proteins have antigen-binding activity against EGFRvIII.

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

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%).

Mannose-Binding Activity of Escherichia coli: a Determinant of Attachment and Ingestion of the Bacteria by Macrophages

1980 ◽  
Vol 29 (2) ◽  
pp. 417-424
Author(s):  
Zvi Bar-Shavit ◽  
Rachel Goldman ◽  
Itzhak Ofek ◽  
Nathan Sharon ◽  
David Mirelman
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Pathogenic Bacteria ◽  
Binding Activity ◽  
Exponential Phase ◽  
Restrictive Temperature ◽  
Filamentous Bacteria ◽  
Phagocytic Cells ◽  
E Coli ◽  
Mannose Binding

Recently, it was suggested that a mannose-specific lectin on the bacterial cell surface is responsible for the recognition by phagocytic cells of certain nonopsonized Escherichia coli strains. In this study we assessed the interaction of two strains of E. coli at different phases of growth with a monolayer of mouse peritoneal macrophages and developed a direct method with [ 14 C]mannan to quantitate the bacterial mannose-binding activity. Normal-sized bacteria were obtained from logarithmic and stationary phases of growth. Nonseptated filamentous cells were formed by growing the organisms in the presence of cephalexin or at a restrictive temperature. Attachment to macrophages of all bacterial forms was inhibited by methyl α- d -mannoside and mannan but not by other sugars tested. The attachment of stationary phase and filamentous bacteria to macrophages, as well as their mannose-binding activity, was similar, whereas in the exponential-phase bacteria they were markedly reduced. The results show a linear relation between the two parameters ( R = 0.98, P < 0.001). The internalization of the filamentous cells attached to macrophages during 45 min of incubation was much less efficient (20%) compared to that of exponential-phase, stationary-phase, or antibody-coated filamentous bacteria (90%). The results indicate that the mannose-binding activity of E. coli determines the recognition of the organisms by phagocytes. They further suggest that administration of β-lactam antibiotics may impair elimination of certain pathogenic bacteria by inducing the formation of filaments which are inefficiently internalized by the host's phagocytic cells.

scholarly journals The Repeat-In-Toxin Family Member TosA Mediates Adherence of Uropathogenic Escherichia coli and Survival during Bacteremia

2011 ◽  
Vol 80 (2) ◽  
pp. 493-505 ◽  
Author(s):  
Patrick D. Vigil ◽  
Travis J. Wiles ◽  
Michael D. Engstrom ◽  
Lev Prasov ◽  
Matthew A. Mulvey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Virulence Factors ◽  
Upper Urinary Tract ◽  
Host Cells ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Novel Target ◽  
Tract Infections

ABSTRACTUropathogenicEscherichia coli(UPEC) is responsible for the majority of uncomplicated urinary tract infections (UTI) and represents the most common bacterial infection in adults. UPEC utilizes a wide range of virulence factors to colonize the host, including the novel repeat-in-toxin (RTX) protein TosA, which is specifically expressed in the host urinary tract and contributes significantly to the virulence and survival of UPEC.tosA, found in strains within the B2 phylogenetic subgroup ofE. coli, serves as a marker for strains that also contain a large number of well-characterized UPEC virulence factors. The presence oftosAin anE. coliisolate predicts successful colonization of the murine model of ascending UTI, regardless of the source of the isolate. Here, a detailed analysis of the function oftosArevealed that this gene is transcriptionally linked to genes encoding a conserved type 1 secretion system similar to other RTX family members. TosA localized to the cell surface and was found to mediate (i) adherence to host cells derived from the upper urinary tract and (ii) survival in disseminated infections and (iii) to enhance lethality during sepsis (as assessed in two different animal models of infection). An experimental vaccine, using purified TosA, protected vaccinated animals against urosepsis. From this work, it was concluded that TosA belongs to a novel group of RTX proteins that mediate adherence and host damage during UTI and urosepsis and could be a novel target for the development of therapeutics to treat ascending UTIs.

scholarly journals Níveis de sensibilidade de enterobactérias, em particular Salmonella typhi, a rifamicina S.V.

1969 ◽  
Vol 3 (2) ◽  
pp. 87-93
Author(s):  
Ivone R. Suassuna ◽  
I. Suassuna ◽  
C. E. de V. Serpa
Keyword(s):  
Escherichia Coli ◽  
Proteus Mirabilis ◽  
Salmonella Typhi ◽  
E Coli

Em face à predominante eliminação biliar da rifamicina S.V. atingindo concentrações muitas vêzes superiores aos níveis séricos obtidos com as doses terapêuticas, e pelo possível interêsse dessa verificação para o tratamento dos portadores biliares crônicos de Salmonella typhi determinou-se a concentração mínima inibitória de 165 estirpes de enterobactérias, incluindo 77 amostras de S. typhi. Foi verificado que a maioria das cepas de Escherichia coli, Shigella e Proteus mirabilis correspondiam a uma concentração inibitória mínima entre 33 a 65 μg/ml. Entre 65 e 128 μg/ml foram determinadas as concetrações inibitórias mínimas da maioria das outras espécies de Proteus, de Providencia e de Klebsiella. Para Salmonella e Enterobacter o limite mínino de sensibilidade foi, em regra, igual ou superior a 128 μg/ml. Diferenças mais acentuadas de comportamento entre as enterobactérias foram observadas quanto à ação bactericida da rifamicidas S.V. De uma maneira geral, para E. coli e Shigella, as concentrações inibitórias mínimas já referidas. Para as espécies de Proteus e Providencia houve variação maior de comportamento, mas tendência a que o efeito bactericidas fôsse encontrado em concentrações que correspondiam a 4 vêzes as bacteriostáticas para as mesmas espécies. Finalmente, de modo pouco feliz para os propósitos visados, em Salmonella, com a inclusão de S. typhi, não foi atingido um efeito bactericida, com as mais altas concentrações usadas as quais corresponderam em média a 6 vêzes as concentrações bacteriostáticas para esse gênero.

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