Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB - E. coli cytoplasm
1 School of Life Sciences, University of Westminster, 115 New Cavendish St, London, W1W 6UW, UK
2 Breast Cancer Biology Group, King's College London, School of Medicine Guy's Hospital, London, SE1 9RT, UK
3 Cardiff University, School of Medicine, Section of Rheumatology, Tenovus Building, Heath Park, Cardiff, CF14 4XN, UK
4 Department of Internal Medicine, University of New Mexico School of Medicine and VA Health Care System,1501 San Pedro Ave SE, Albuquerque, NM 87108, USA
5 Queen Mary University of London, Barts & The London School of Medicine and Dentistry, Institute of Dentistry, 4 Newark Street, London, E1 2AT, UK
BMC Biotechnology 2011, 11:117 doi:10.1186/1472-6750-11-117Published: 30 November 2011
Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains linked by long flexible linkers are themselves prone to disassociation and aggregation, and in some cases with isoelectric points incompatible with use in physiologically relevant milieu. Here we describe a general approach that permits fully functional intracellular production of a range of coloured fluorescent recombinant antibodies with optimally orientated VH/VL interfaces and isoelectric points compatible for use in physiological solutions at pH 7.4 with a binding site to fluorophore stoichiometry of 1:1.
Here we report the design, assembly, intracellular bacterial production and purification of a panel of novel antibody fluorescent protein fusion constructs. The insertion of monomeric fluorescent protein derived from either Discosoma or Aequorea in-between the variable regions of anti-p185HER2-ECD antibody 4D5-8 resulted in optimal VH/VL interface interactions to create soluble coloured antibodies each with a single binding site, with isoelectric points of 6.5- 6. The fluorescent antibodies used in cell staining studies with SK-BR-3 cells retained the fluorophore properties and antibody specificity functions, whereas the conventional 4D5-8 single chain antibody with a (Gly4Ser)3 linker precipitated at physiological pH 7.4.
This modular monomeric recombinant fluorescent antibody platform may be used to create a range of recombinant coloured antibody molecules for quantitative in situ, in vivo and ex vivo imaging, cell sorting and cell trafficking studies. Assembling the single chain antibody with monomeric fluorescent protein linker facilitates optimal variable domain pairing and alters the isoelectric point of the recombinant 4D5-8 protein conferring solubility at physiological pH 7.4. The efficient intracellular expression of these functional molecules opens up the possibility of developing an alternative approach for tagging intracellular targets with fluorescent proteins for a range of molecular cell biology imaging studies.