Expression of anti-Z-DNA single chain antibody variable fragment on the filamentous phage surface

Abstract

We describe the expression of an anti-Z-DNA single chain variable region antibody fragment (scFv) on a filamentous phage surface. Four vectors for phage display were constructed. Two of them are able to display multiple copies of the antibody fragment, and the others can be used to make monovalent libraries. The vectors use different promoter/leader sequences to direct the expression of the fused proteins. All were able to promote the assembly of fusion virion particles. In this paper we also show the affinity selection (biopanning) of those phageantibodies based on the capacity of their products to recognize the antigen. We used biotinylated Z-DNA and the selection was performed in a solution phase fashion. The data presented here indicate that these vectors can be further used to construct anti-nucleic acid antibody fragment libraries that can be used to study the basis of nucleic acid-protein interaction and its role in autoimmunity mechanisms.

A Mouse Variable Gene Fragment Binds to DNA Independently of the BCR Context: A Possible Role for Immature B-Cell Repertoire Establishment

“…Hence, a V10-bearing Fv should be less dependent on both the HCDR3 and the light chain. An experimental system was developed based on the pCIg816 phage display vector [20], a phagemid created to obtain a scFv fragment fused to the M13 gene 8 on the bacteriophage capsid. In this vector, we introduced either a V4 or a V10 germline sequence along with a library of 9 mer HCDR3 and a JH4 gene fragment forming the variable heavy chain, concatenated with a fixed V chain of the Z44 antibody [21].…”

“…Either a V10 or V4 germline V gene fragment was transferred to the pCIg 816 phagemid vector [20]. This plasmid codes for a scFv (V linker V) fused to the M13 gene 8 at its carboxi terminus.…”

Antibody Phage Display Libraries: Contributions to Oncology

“…Although well established, this technology is laborious, and it is biased by the experimental animal immune system, which limits the ability to reach a high-affinity antibody against conserved mammal proteins. Additionally, the heterologous character of those proteins turn them often immunogenic to humans eliciting HAMA response (Human Anti-Mouse Antibodies), which restrict their therapeutic use [5]. Human antibodies are of particular interest due to a lower immunogenicity response [1].…”

“…For this aim, primers covering all V gene families are used and the library is generated by a random combination of VL and VH chain coding genes. Again, the antibody coding genes are assembled to be displayed as Fab or scFv antibody fragments [5]. Theoretically, each clone codes for a specific antigen-binding site, derived from the natural repertoire which is increased by an artificial domain shuffling that extrapolates the original repertoire.…”

Método in silico para análise de sequências de imunoglobulinas produzidas por tecnologia de phage display

“…O gene da proteína de interesseé fusionado ao gene de uma proteína do capsídeo do fago, o qual infecta a célula bacteriana e assim, assegura-se a expressão da proteína de estudo durante a etapa de produção de proteínas essenciaisà montagem da partícula viral. Desse modo, uma biblioteca de genes de interesse, por exemplo, genes codificantes de fragmentos de anticorpos recombinantes,é gerada utilizando como veículo de expressão o genoma de fagos filamentosos (Maranhão & Brígido, 2000;Willats, 2002;Walsh, 2007).…”