(prokaryotic expression vector/chloramphenicol-affinity purification)

Sequence studies of the adenovirus 2 genome have revealed the presence of a large open reading frame (ORF) from 22.9 to 14.2 map units that is believed to encode most of the adenovirus DNA polymerase (Ad Pol). An 838- base-pair fragment (19.6-17.3 map units) containing approxi- mately 25 % of this ORF has been cloned and expressed in a ft- galactosidase-chloramphenicol acetyltransferase (lacZ-CAT) expression vector under the control of the trp-lac hybrid pro- moter. This recombinant vector directed the synthesis of a 58- kDa lacZ-Ad Pol-CAT fusion protein that has CAT activity. This fusion protein was easily purified lby affinity chromatog- raphy in which chloramphenicol, the substrate for CAT, was covalently bound to a matrix. Antisera were prepared against the purified 58-kDa lacZ-Ad Pol-CAT fusion protein and were found to react specifically with the 140-kDa Ad Pol by ELISA and immunoblot analysis. In addition, these antisera recog- nized 120- and 29-kDa polypeptides in immunoblot analysis of partially purified terminal protein precursor (pTP)-Ad Pol complex. The exact nature of the 120- and 29-kDa polypep- tides is not known, but they may be breakdown products of Ad Pol. Although the lacZ-Ad Pol-CAT fusion protein is not active in any of the Ad Pol enzymatic reactions, antibody against the prokaryotic fusion protein should be useful for screening bac- teria harboring plasmids that have been constructed to express the entire Ad Pol ORF.