Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB- E. colicytoplasm

Anatoliy Markiv,Joy Burchell,Angray S Kang,Ravi V Durvasula,Richard Beatson

doi:10.1186/1472-6750-11-117

Abstract

BackgroundAntibody-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.ResultsHere 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.ConclusionsThis 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.

Highlights

Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events
Molecular modelling A rational approach was used in engineering of single-chain fragment variable (scFv) 4D5-8 fusion with mRFP1 based on the x-ray crystal structure of 4D5-8 Fab (PDB 1N8Z) [46] and the modelled structure of mRFP1 based on the DsRed dimer
This precise spatial geometry would permit the docking of mRFP1 or any of its derivatives between the antibody VH/VL or VL/ VH chains resulting in functional scFv fluorophore fusions

Summary

Introduction

Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. Conventional scFv antibody domains linked by long flexible linkers are themselves prone to dissociation and aggregation [20,21], reducing the specific activity and fourthly the requirement for secretion of the recombinant antibodies into the oxidising periplasmic space permitting intra molecular disulfide bond formation significantly reduces the yield (0.10.2 mg of antibody-GFP fusion/L bacterial culture) [22]. Alternative expression platforms such as mammalian and insect cells have been used to produce scFvGFP fusions [23,24,25], but at increased costs. These alternative expression modalities dealt with increasing the recovery of the secreted recombinant molecules and not the intrinsic stability of the antibody-GFP proteins

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