Previous experiments were designed to test the assumption that the conserved residues (CR) of a protein are essential for its function. Procedures were developed for efficient use of the polymerase chain reaction (PCR) to introduce alterations into codons for CRs in a region of the vaccinia virus DNA polymerase. These alterations are linked to a mutation in an adjacent site which confers resistance to aphidicolin (AP). The most significant substitutions were the replacement of a pair of amino acids, tyrosine and valine, by either tryptophan and threonine or tryptophan and alanine in the same sequence. Vaccinia virus with these alterations produced normal virus. Attempts to replace the same tyrosine with aspartic acid failed, indicating that a negative charge could not be introduced in this region. Also, attempts to replace the conserved lysine were not successful. To test these in vivo changes, I have tried to develop a bacterial cloning and expression system. To obtain more faithful PCR replication of target DNA, I have replaced Taq polymerase with Vent polymerase and modified the reaction conditions. To facilitate cloning procedures, I developed a PCR assay which replaces the """"""""mini prep"""""""" procedure for detecting the presence and orientation of inserted DNA. Finally, I modified the affinity resin binding procedure used with pET vectors so that it could be used with detergent-solubilized proteins. Using these procedures, I tried to express a fusion protein which included the carboxy terminal portion of the DNA polymerase. Use of the pMal vector to produce a fusion with the target led to degradation of the fused protein. A pET vector produces a protein of the predicted size in amounts that can be detected by stained gels. If the protein is genuine and can be renatured, it should be suitable for in vitro studies.
