High titer expression of antibodies using linear expression cassettes for early-stage functional screening.
Protein Eng Des Sel
; 372024 Jan 29.
Article
em En
| MEDLINE
| ID: mdl-39141844
ABSTRACT
Antibody discovery processes are continually advancing, with an ever-increasing number of potential binding sequences being identified out of in vivo, in vitro, and in silico sources. In this work we describe a rapid system for high yield recombinant antibody (IgG and Fab) expression using Gibson assembled linear DNA fragments (GLFs). The purified recombinant antibody yields from 1 ml expression for this process are approximately five to ten-fold higher than previous methods, largely due to novel usage of protecting flanking sequences on the 5' and 3' ends of the GLF. This method is adaptable for small scale (1 ml) expression and purification for rapid evaluation of binding and activity, in addition to larger scales (30 ml) for more sensitive assays requiring milligram quantities of antibody purified over two columns (Protein A and size exclusion chromatography). When compared to plasmid-based expression, these methods provide nearly equivalent yield of high-quality material across multiple applications, allowing for reduced costs and turnaround times to enhance the antibody discovery process.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes
/
Imunoglobulina G
/
Fragmentos Fab das Imunoglobulinas
Limite:
Humans
Idioma:
En
Revista:
Protein Eng Des Sel
Assunto da revista:
BIOQUIMICA
/
BIOTECNOLOGIA
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Estados Unidos
País de publicação:
ENGLAND
/
ESCOCIA
/
GB
/
GREAT BRITAIN
/
INGLATERRA
/
REINO UNIDO
/
SCOTLAND
/
UK
/
UNITED KINGDOM