Construction, characterisation and utilisation of a novel E. coli expression vector

At the onset of this thesis the aim was to design and develop an expression system capable of producing high levels of expression of carboxypeptidase (CPG2). The previous plasmid employed (pNM21) delivered relatively low and variable levels of CPG2. Plasmid segregational instability had been thought to be a potential cause of low yields of CPG2. The effect of the par locus on the stability of a series of related plasmids was examined. The utilisation of par within a vector did enhance its stability and fitness when examined under continuous cultivation. The par locus was therefore considered to be of significant value to an expression vector. The par locus subsequently became an integral feature incorporated into any vector to be constructed. The copy numbers of the series of plasmids examined for stability revealed that pUC8 exhibited a higher copy number than pAT153. To investigate more fully this anomaly, a vector was constructed which provided the backbone for a series of cloning vectors (the pMTL series) and allowed the copy number variation to be examined. Sequencing pAT153 and pUC8 revealed a single base change, which could account for the copy number variation. The increase in copy number of pl) C8 over pAT153 was incorporated in the pMTL series of vectors. The induced expression of CPG2 was investigated using the Pl promoter on a high copy number vector, exhibiting the aforementioned base change. Incomplete repression of the Pl promoter resulted when the c_I gene was located in the chromosome or on a co-resident low copy number vector. The high copy number of the vector and the Pl operator it carried, titrates out the effectiveness of the cl repressor molecule. This problem was overcome by incorporation of the £l gene into the Pl expression vector, elevating the gene dosage of the cl_ gene, sufficient to repress the Pl promoter. The features investigated were combined in an expression vector pCMIO and used to express CPG2 in a series of small scale fermentations. The process was then scaled up to a 400 L fermentation, and a suitable process for large scale production developed