Isolation and characterisation of monoclonal antibodies raised against the Pk peptide tag

Abstract

In this project, a number of monoclonal antibodies were developed which recognised an oligopeptide tag known as the Pk tag. These antibodies were used for the development of an affinity purification protocol in order to purify Pk tagged recombinant proteins. These proteins, in turn, were to be used for the development of multiple epitope vaccines. The Pk tag is a 14 amino acid oligopeptide which was originally mapped to the binding epitope of the monoclonal antibody SV5-P-k, raised to the Paramyxovirus Simian Virus 5 (SV5) phospho- (P) protein. The tag has been cloned into the bacterial expression vector pQ9cPk so that when expressed, any recombinant protein produced will have a Histidine tag at the N terminus and the Pk tag at the C terminus. These two tags are the basis of a two step purification system, utilising a nickel affinity column for the Histidine tag capture, and monoclonal antibody (mAb) SV5-P-k immobilised on Sepharose beads for Pk tag capture. The genes encoding the Simian Immunodeficiency Virus (SIV) precursor protein Pr55gag and the auxiliary protein Nef were cloned into pQ9cPk and the recombinant proteins produced purified using the current two step purification protocol. However, the affinity of the mAb SV5-P-k for the Pk tag is so strong, that denaturing conditions were needed to separate the antibody from the tagged protein. Due to this it was decided to develop a number of mAb's to the Pk tag with the aim of creating antibodies whose binding affinities varied from that of SV5-P-k. A number of hybridoma clones expressing mAb's which recognise the Pk tag were created and after initial characterisation it was demonstrated that of the twenty four clones produced, only four recognised Pk tagged recombinant proteins or the SV5 P protein. The epitopes and binding affinities of these four mAb's were determined, and when compared to SV5-P-k, it was observed that all five mAb's had similar affinities for the Pk tag. Therefore, antibodies to the Pk tag which had lower binding affinities were not obtained. In order to utilise the five anti-Pk mAb's in the two step purification system, the original primary sequence of the Pk tag was modified, creating three new tags. On further analysis of the binding of the five anti-Pk mAb's to the modified tags, elution conditions were determined which allowed the elution of Pk tagged recombinant proteins from a mAb bound affinity column.