Generation of Fluorescent Protein-single Chain Antibody Fusion Products as Bio-detection and Identification Reagents

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Authors
  1. Jager, S.
  2. Pon, J.K.
  3. Mah, M.
  4. Mah, D.
  5. Fulton, R.E.
Corporate Authors
Defence Research Establishment Suffield, Ralston ALTA (CAN);Canada West Biosciences, Calgary ALTA (CAN)
Abstract
We have genetically fused an the gene encoding recombinant (GFPuv to the 3' end of anti-BoNT/A D12 scFv antibody gene in expression vector pRSD12-8. This scFv-GFP fusion vector which is under the control of the T7 promoter expresses the D12-GFPuv fusion protein (60.6kDa) as non-fluorescent inclusion bodes. Inclusion bodies can be denature in 8 M urea and purified on IMAC (immobilized metal affinity chromatography) columns, but attempts at renaturation failed to regenerate a fluorescent fusion product. We hypothesize that th eloss of fluorescence is due the improper folding which occurs as a result of high-level expression and/or misguiding of protein folding by the preceding scFv polypeptide during protein synthesis. Work was initiated to construct an GFP-scFv fusion expression vector under the control of the lacZ promoter to test these hypotehsis. This contract delivers a plasmid DNA and/or bacterial glycerol stocks of T7 promoter-based scFv-GFP fusion expression vectors (pRSD12guv-1 or pRSD12guv-2) and intermiedate plasmid constructs for a GFP-scFv fusion expression vector (pGuvD12). In addition, seventeen (17) anti-ovalbumin mouse monoclonal hybridoma cell lines (derived from 5 primary clones) are delivered along with one anti-ovalbumin scFv clone (p12H6H8F3) in phage-display vector pCantab5E. TRUNCATED
Keywords
Fusion antibodies;Immunodetection reagents;Biosensors
Report Number
DRES-CR-1999-142;EDM-6-00625 — Contractor Report (Final)
Date of publication
01 Nov 1999
Number of Pages
122
DSTKIM No
CA000095
CANDIS No
512210
Format(s):
Hardcopy;Document Image stored on Optical Disk

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