Bacterial expression of anti-DNA antibody domains

Methods : a Companion to Methods in Enzymology
B D Stollar

Abstract

Bacterial production of recombinant Fab or Fv domains of antibodies is an important tool for analyzing structural correlates of antigen binding or idiotype expression. Bacterial products may be Fab or Fv molecules that assemble from separate chains in the periplasm or a single-chain Fv protein. This article describes properties and applications of a plasmid vector used for production of single-chain Fv (scFv). The expression cassette, initially designed for production of the Fv domain of anti-Z-DNA mAb Z22, has a bacterial secretion signal that permits secretion of soluble scFv into growth medium. A single B domain of staphylococcal protein A facilitates affinity purification and generic assay of products independent of antigen-binding activity. Gene segment swapping and directed mutagenesis identified structural features important for Z-DNA binding and revealed the structural similarity of autoantibody and immunization-induced antibody. The H and L regions of mAb Z22 are readily replaced by those of any cloned Ig, a library of V regions, or other proteins. Modified forms of the vector code for production of separate H or L chain V regions, which can associate with each other to form functional Fv complexes. Although there is l...Continue Reading

Citations

Apr 29, 1999·Proceedings of the National Academy of Sciences of the United States of America·G R RosaniaR Heald
Dec 31, 1997·The Journal of Cell Biology·S S Andersen, E Karsenti
Oct 11, 2005·Biochimica Et Biophysica Acta·Edmar Vaz de AndradeMarcelo Macedo Brigido
Feb 17, 2001·Journal of Molecular Recognition : JMR·K C O'ConnorB D Stollar
Jan 17, 2003·Lupus·S KumarD A Isenberg
May 10, 2000·The Journal of Biological Chemistry·H L FordD C Seldin

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.