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Title: Cloning and expression of a human type V acid phosphatase single chain antibody fragment in Escherichia coli and Pichia pastoris
Author: Cupit, Pauline M.
ISNI:       0000 0001 3400 5107
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1996
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A recombinant single chain antibody (scAb) fragment specific for the isoenzyme human type V acid phosphatase has been expressed and secreted from Escherichia coli at a level of 0.02 mg/litre culture volume. Purification of the fragment using antibody immunoaffinity and metal chelate affinity chromatography yielded a mixture of dimer and monomer. The binding sensitivity of the dimeric scAb was ten fold lower than that of the parent monoclonal antibody as determined by BIAcore and ELISA. The methylotrophic yeast Pichia pastoris was used for high level expression of the scAb fragment. Using the P. pastoris expression vector pHIL-S1, the yield of scAb was 0.44 mg/litre culture volume, however, the fragment did not bind the target antigen as well as the scAb expressed from E. coli. More than 2.1 mg/litre culture volume of the scAb was secreted from the P. pastoris vector pPIC9 but the fragment was not functional.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Antibodies Molecular biology Cytology Genetics Microbiology