A simple vector system to improve performance and utilisation of recombinant antibodies.
BACKGROUND
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. 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.


RESULTS
We 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.


CONCLUSIONS
Nine 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.
Martin
Cecile D
CD
Atlas of Protein Expression, Wellcome Trust Sanger Institute, Genome Campus, Morgan Building, Hinxton, Cambridgeshire, CB10 1HH, UK. cecile.d.martin@gsk.com <cecile.d.martin@gsk.com>
Rojas
Gertrudis
G
Mitchell
Joanne N
JN
Vincent
Karen J
KJ
Wu
Jiahua
J
McCafferty
John
J
Schofield
Darren J
DJ
D000469:Alkaline Phosphatase / Q000235:genetics; D015153:Blotting, Western; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D007118:Immunoassay; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D007150:Immunohistochemistry; D019151:Peptide Library; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D014764:Viral Proteins
D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D000469:Alkaline Phosphatase