scFv Antibody Fragment Expression and Purification

What is scFv Antibody

A single-chain fragment variable (scFv) is a recombinant antibody and has great potential for use in many fields such as research, diagnostic and clinical applications. The molecular weight is approximately 28 kDa. It consists of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of 10-25 amino acids. Each VH and VL domain contains three complementarity determining regions (CDRs). CDRs are short amino acid sequences that vary greatly among antibody molecules and, thus, are responsible for generating the great diversity of antibody binding specificity. The combination of the CDRs of the VH plus the CDRs of the VL determines the binding specificity of any given antibody.

Structure of scFv antibody fragment
Fig 1. Structure of scFv antibody fragment

Production of Anti-scFv Antibody

Phage display technology has greatly facilitated the genetic manipulation of antibody fragments and has led the development of a large variety of engineered antibody molecules for research, diagnosis, and therapy with specificities out of reach of conventional antibody technology. Once cloned, it is then possible to increase the affinity and specificity of antigen binding by mimicking somatic hypermutation during an immune response. It may even be possible to replace the existing practices of animal immunization and hybridoma development through a bacterial system capable of synthesizing and expressing practically unlimited quantities of antibodies to almost any antigen.

In order to get scFv antibody fragments, mRNA is first isolated from hybridoma (or spleen, lymph cells, and bone morrow) followed by reverse transcribed into cDNA to serve as a template for antibody genes amplification (PCR). With this technique, large libraries with a diverse range of antibody VH and VL genes could be created. The biopanning step helps us get the scFv antibody fragment with the best affinity and specificity. In the construction of scFv, the order of the domains can be either VH-linker-VL or VL-linker-VH and both orientations have been applied.

scFv antibody fragment expression and production is a routine work to support antibody discovery with antibody library. When the antibody genes were successfully cloned and sequenced, single chain Fv fragments can be readily expressed in microbial expression system such as E. coli. It can also be expressed in mammalian systems (i.e., HEK293 cells) through transient transfection. Sino Biological is capable of scFv antibody fragment generation using either production systems, depending on customer's preference.

A suitable purification tag is typically added to the C-terminus of the antibody scFv fragment protein. Commonly used purification tags are ploy-histidine tag, FLAG-tag, HA-tag, and Myc-tag. An protease cleavage size can be designed between the His-tag and the Fab to allow tag-removal after purification.

Construction and production details about our scFv fragment antibody service

Host cells E. coli., Yeast, or HEK293
Production Recombinant technology
- E. coli fermentation: soluble expression
- Yeast Fermentation: secrected protein expression
- Mammalian cell transient transfection
Purification method • His-tag, FLAG-tag etc. Affinity chromatography column purification
• Anion and Caton Exchange Chromatography
• and Size Exclusion Chromatography Column (SEC) for polishing if needed
Purification tags 6-10 His tag, FLAG tag, SUMO-tag, etc. as chosen by customer
Timeline 3-6 weeks total from receiving gene sequence to product delivery
Deliverable Purified scFv antibody (quantities to be specified by customer)
Quality specification as specified by customer (cost may be adjusted based on quality specification)
Antibody types Human, mouse, rat, rabbit, canine, rhesus IgG antibodies
Applications Drug discovery: antibody antigen co-crystalization, antibody binding epitope


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4. Tue-Ngeun, P., Kodchakorn, K., Nimmanpipug, P., Lawan, N., Nangola, S., Tayapiwatana, C., ... & Lee, V. S. (2013). Improved SCFV ANTI-HIV-1 P17 binding affinity guided from the theoretical calculation of pairwise decomposition energies and Computational Alanine Scanning. BioMed research international, 2013.

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