Fox, Simon George; (1999) Expression of catalytic antibody C3 esterase scFv in Escherichia coli. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes the construction of a vector for the expression of an antibody light chain variable region gene, (V1), from the monoclonal catalytic antibody C3 in Escherichia coli. The antibody C3 was developed by Khalaf, Suckling, Stimson et al (1992) at the University of Strathclyde, and catalysed the hydrolysis of 4-nitrophenyl 5-(3-methoxyphenyl)-pentanoate. The binding site of the C3 abzyme was cloned as an scFv by the University of Leicester into an expression vector pPANG1 developed at Leicester. The VH sequence in the C3scFv construct was found to be an aberrantly rearranged myeloma VH sequence, and not the genuine C3 VH sequence. Expression of the C3 scFv sequence from pPANG1 failed to produce any protein. The genuine C3 V1 sequence was cloned into a novel expression vector, pQR627, based on pBluescript II. The novel expression vector carried the C3V1 gene as a fusion to a antibody binding domain of Staphylococcus aureus protein A. The C3V1 gene was expressed in E.coli from vector pQR627. The results of the expression experiments with vector pQR627 indicated that the C3V1 gene product was cytotoxic to the host E.coli cells. This cytotoxicity was the probable cause of the low expression titre of the C3V1 gene product in pQR627 cultures. The SpA- C3V1 protein was probed for determination of its binding properties and its catalytic properties. The SpA- C3V1 failed to exhibit the binding properties of the parent monoclonal antibody C3, it did however exhibit a catalytic function. The SpA- C3V1 protein was found to have 5.9% of the specific activity of the whole C3 IgG as determined by Khalaf, Suckling, Stimson et al (1992).

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