Expression of mouse immunoglobulin light and heavy chain variable regions in Escherichia coli and reconstitution of antigen-binding activity

1990 ◽  
Vol 3 (7) ◽  
pp. 641-647 ◽  
Author(s):  
Helen Field ◽  
G.T. Yarranton ◽  
A.R. Rees
Keyword(s):  
Escherichia Coli ◽  
Heavy Chain ◽  
Binding Activity ◽  
Antigen Binding ◽  
Variable Regions ◽  
Mouse Immunoglobulin

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 Systematic Exploration of the Antigen Binding Activity of Synthetic Peptides Isolated from the Variable Regions of Immunoglobulins

1997 ◽  
Vol 272 (49) ◽  
pp. 30937-30944 ◽  
Author(s):  
Daniel Laune ◽  
Franck Molina ◽  
Gaelle Ferrieres ◽  
Jean-Claude Mani ◽  
Pascale Cohen ◽  
...  
Keyword(s):  
Synthetic Peptides ◽  
Binding Activity ◽  
Antigen Binding ◽  
Variable Regions ◽  
Systematic Exploration

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.

scholarly journals Multidrug Resistance Dissemination in Escherichia coli Isolated from Wild Animals: Bacterial Clones and Plasmid Complicity

2021 ◽  
Vol 12 (1) ◽  
pp. 123-137
Author(s):  
Carolina Sabença ◽  
Gilberto Igrejas ◽  
Patrícia Poeta ◽  
Frédéric Robin ◽  
Richard Bonnet ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epidemiological Data ◽  
Generation Cephalosporin ◽  
Wild Animals ◽  
Variable Regions ◽  
E Coli ◽  
Wild Fauna ◽  
Long Read ◽  
Sequence Types ◽  
Animal Isolates

Objectives. Epidemiological data concerning third-generation cephalosporin (3GC) resistance in wild fauna are scarce. The aim of this study was to characterize the resistance genes, their genetic context, and clonal relatedness in 17 Escherichia coli resistant to 3GC isolated from wild animals. Methods. The isolates were characterized by short-read whole genome sequencing, and long-read sequencing was used for the hybrid assembly of plasmid sequences. Results. The 3GC resistance gene most identified in the isolates was the extended-spectrum β-lactamases (ESBL)-encoding gene blaCTX-M-1 (82.3%), followed by blaCTX-M-32 (5.9%), blaCTX-M-14 (5.9%), and blaSHV-12 (5.9%). E. coli isolates mainly belonged to the sequence types (STs) rarely reported from humans. The single nucleotide polymorphism (SNP)-based typing showed that most E. coli genomes from wild animals (wild boars, birds of prey, and buzzards) formed clonal clusters (<5 SNPs), showing a clonal dissemination crossing species boundaries. blaCTX-M-1-harboring IncI1-ST3 plasmid was the predominant ESBL-encoding plasmid (76.4%) in wild animal isolates. Plasmid comparison revealed a 110-kb self-transferable plasmid consisting of a conserved backbone and two variable regions involved in antimicrobial resistance and in interaction with recipient cells during conjugation. Conclusion. Our results highlighted the unexpected clonal dissemination of blaCTX-M-1-encoding clones and the complicity of IncI1-ST3 plasmid in the spread of blaCTX-M-1 within wild fauna.

scholarly journals Human rheumatoid factor crossidiotypes. I. WA and BLA are heat-labile conformational antigens requiring both heavy and light chains.

1986 ◽  
Vol 164 (5) ◽  
pp. 1809-1814 ◽  
Author(s):  
V Agnello ◽  
J L Barnes
Keyword(s):  
Rheumatoid Factor ◽  
Primary Structure ◽  
Binding Activity ◽  
Light Chains ◽  
Heat Denaturation ◽  
Antigen Binding ◽  
Rheumatoid Factors ◽  
Simple Method ◽  
Heat Labile

Evidence was obtained that both the WA and BLA crossidiotype (XId) groups are conformational antigens requiring both L and H chains and that with heat denaturation the antigens that define the XIds and antigen-binding activity are lost in parallel. In contrast, the primary structure-dependent crossreactive idiotype (CRI), PSL2, which is only weakly detected on native Wa and Bla monoclonal rheumatoid factors (mRFs), became prominently detected on the heated Wa and Bla mRFs. Heat denaturation may provide a simple method for distinguishing Ids determined by conformational antigen from primary structure-dependent Ids. In addition to heat denaturation, some acid conditions commonly used for preparation of RFs were also found to cause marked loss of Id antigen. The finding of PSL2-CRI on Bla mRF indicates that this Id is not unique to the WA XId.

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