Production of α-Synuclein Fibrillar-Specific scFv from Inclusion Bodies.

Recombinant antibody fragments such as Fab, scFvs, and diabodies against α-syn have become a viable alternative to the conventional full-length antibodies in immunotherapeutic approaches due to their benefits which include smaller size, higher stability, specificity, and affinity. However, the majority of recombinant antibody fragments typically express as inclusion bodies (IBs) in E. coli, which makes their purification incredibly difficult. Here, we describe a method involving a mild solubilizing protocol followed by slow on-column refolding to purify active single-chain variable fragment (scFv-pF) antibody that can recognize the pathogenic α-syn fibrils.

Gupta V ，Hmila I ，Vaikath NN ，Sudhakaran IP ，El-Agnaf OMA ... -  《-》

Expression, purification and characterization of α-synuclein fibrillar specific scFv from inclusion bodies.

Gupta V ，Sudhakaran IP ，Islam Z ，Vaikath NN ，Hmila I ，Lukacsovich T ，Kolatkar PR ，El-Agnaf OMA ... -  《PLoS One》

Fibrillar form of α-synuclein-specific scFv antibody inhibits α-synuclein seeds induced aggregation and toxicity.

Gupta V ，Salim S ，Hmila I ，Vaikath NN ，Sudhakaran IP ，Ghanem SS ，Majbour NK ，Abdulla SA ，Emara MM ，Abdesselem HB ，Lukacsovich T ，Erskine D ，El-Agnaf OMA ... -  《Scientific Reports》

Blocking monocyte transmigration in in vitro system by a human antibody scFv anti-CD99. Efficient large scale purification from periplasmic inclusion bodies in E. coli expression system.

Migration of leukocytes into site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells, inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies and the absence of toxic reagents utilized for solubilization and refolding step of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting, we herein describe an efficient and large scale production of the antibody fragments expressed in E. coli as periplasmic insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signaling. This protocol can be useful for the successful purification of other monomeric scFvs which are expressed as periplasmic inclusion bodies in bacterial systems.

Moricoli D ，Muller WA ，Carbonella DC ，Balducci MC ，Dominici S ，Watson R ，Fiori V ，Weber E ，Cianfriglia M ，Scotlandi K ，Magnani M ... -  《-》

Production of Stabilized Antibody Fragments in the E. coli Bacterial Cytoplasm and in Transiently Transfected Mammalian Cells.

Birnboim-Perach R ，Grinberg Y ，Vaks L ，Nahary L ，Benhar I ... -  《-》