Effects of variable domain orientation on anti‐HER2 single‐chain variable fragment antibody expressed in the Escherichia coli cytoplasm

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.

Engineering of a Single-Chain Variable-Fragment (scFv) Antibody Specific for the Stolbur Phytoplasma (Mollicute) and Its Expression in Escherichia coli and Tobacco Plants

Applied and Environmental Microbiology ◽

10.1128/aem.64.11.4566-4572.1998 ◽

1998 ◽

Vol 64 (11) ◽

pp. 4566-4572 ◽

Cited By ~ 45

Author(s):

Fabrice Le Gall ◽

Joseph-Marie Bové ◽

Monique Garnier

Keyword(s):

Escherichia Coli ◽

Transgenic Tobacco ◽

Enzyme Linked Immunosorbent Assay ◽

Bacterial Cells ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Tobacco Plants ◽

Challenge Inoculation ◽

Scfv Gene ◽

Stolbur Phytoplasma

From a hybridoma cell line (2A10) producing an immunoglobulin G1 directed against the major membrane protein of the stolbur phytoplasma, we have engineered scFv (single-chain variable-fragment) antibodies from the variable heavy (VH) and light (VL) domains of the immunoglobulin. The scFv gene was cloned and expressed inEscherichia coli. The expressed protein of 30 kDa could be recovered from the periplasmic fraction of the bacterial cells and was shown to be fully functional toward its phytoplasmal antigen, since enzyme-linked immunosorbent assay or immunofluorescence (IF) detection of the stolbur phytoplasma antigen by the scFv was identical to that of the native immunoglobulin. The scFv gene was then cloned in plasmid pBG-dAb-BIN of Agrobacterium tumefaciens to transform tobacco plants. The transformed plants were screened by PCR and Northern blotting for the presence and expression of the transgene, respectively, and by IF for expression of the scFv. One transgenic tobacco line, 1A6, was selected for challenge inoculation with the stolbur phytoplasma. When grafted on a stolbur phytoplasma-infected tobacco rootstock, the transgenic tobacco shoots grew free of symptoms and flowered after 2 months, while normal tobacco shoots showed severe stolbur symptoms during the same period and eventually died.

Antigenic epitopes on the outer membrane protein A of Escherichia coli identified with single-chain variable fragment (scFv) antibodies

Applied Microbiology and Biotechnology ◽

10.1007/s00253-019-09761-8 ◽

2019 ◽

Vol 103 (13) ◽

pp. 5285-5299 ◽

Cited By ~ 1

Author(s):

Pharaoh Fellow Mwale ◽

Chi-Hsin Lee ◽

Sy-Jye Leu ◽

Yu-Ching Lee ◽

Hsueh-Hsia Wu ◽

...

Keyword(s):

Escherichia Coli ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Protein A ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Outer Membrane Protein A ◽

Antigenic Epitopes

Cloning, expression and purification of a single chain variable fragment antibody against Her2 in Escherichia coli

New Biotechnology ◽

10.1016/j.nbt.2012.08.327 ◽

2012 ◽

Vol 29 ◽

pp. S117

Author(s):

Vajihe Akbari ◽

Daryoush Abedi ◽

Hamid Mir Mohammad Sadeghi ◽

Abbas Jafrian-Dehkordi ◽

Perry Chou

Keyword(s):

Escherichia Coli ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Expression And Purification