Abstract
BackgroundIsolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression.ResultsWe have developed a simple vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of single chain Fvs (scFvs). Expression is driven by the T7 RNA polymerase promoter in conjunction with the inducible lysogen strain BL21 (DE3). The system is compatible with a simple auto-induction culture system for scFv production. As an alternative to periplasmic expression, expression directly in the cytoplasm of a mutant strain with a more oxidising cytoplasmic environment (Origami 2™ (DE3)) was investigated and found to be inferior to periplasmic expression in BL21 (DE3) cells. The effect on yield and binding activity of fusing scFvs to the N terminus of maltose binding protein (a solubility enhancing partner), bacterial alkaline phosphatase (a naturally dimeric enzymatic reporter molecule), or the addition of a free C-terminal cysteine was determined. Fusion of scFvs to the N-terminus of maltose binding protein increased scFv yield but binding activity of the scFv was compromised. In contrast, fusion to the N-terminus of bacterial alkaline phosphatase led to an improved performance. Alkaline phosphatase provides a convenient tag allowing direct enzymatic detection of scFv fusions within crude extracts without the need for secondary reagents. Alkaline phosphatase also drives dimerisation of the scFv leading to an improvement in performance compared to monovalent constructs. This is illustrated by ELISA, western blot and immunohistochemistry.ConclusionNine scFv expression vectors have been generated and tested. Three vectors showed utility for expression of functional scFv fragments. One vector, pSANG14-3F, produces scFv-alkaline phosphatase fusion molecules which offers a simple, convenient and sensitive way of determining the reactivity of recombinant antibody fragments in a variety of common assay systems.
Highlights
Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display
There were five different single chain Fvs (scFvs) expression forms: scFv only, scFv plus a C-terminal cysteine residue, scFv fused to maltose binding protein, scFv-MBP plus a C-terminal cysteine residue, and scFv fused to bacterial alkaline phosphatase
Additional constructs without the pelB leader sequence were made in order to produce the scFv, scFvcys, scFv-MBP, and scFv-MBP-cys forms expressed in the cytoplasm rather than the periplasmic space in Origami 2TM (DE3) cells
Summary
Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. They are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression. The isolation of recombinant antibody fragments with unique binding specificities can be readily accomplished using antibody display methods such as phage display or ribosome display In such display methods large naïve libraries are generated where the gene encoding an antibody is physically linked to the resulting antibody protein (in the form of a single chain Fv fragment (scFv)). Resultant products are fused with a six histidine tag for one step immobilised metal affinity chromatography (IMAC) and a tri-FLAG epitope tag for detection
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