INVESTIGATIONS ON THE THERMOSTABILITY AND FUNCTION OF TRUNCATED THERMUS-AQUATICUS DNA-POLYMERASE FRAGMENTS

The thermostable DNA polymerase from Thermus aquaticus (Tag polymerase ) has been truncated to molecular regions essential for polymerase act ivity. Two truncated forms of the full-length 832 amino acid Tag polym erase have been constructed according to sequence alignments and the k nown domain structure of the homologous Escherichia coli DNA polymeras e I (E.coli pol I): variant Delta 288 (lacking the N-terminal 288 amin o acid portion) and variant Delta 413 (lacking the N-terminal 413 amin o acid portion), Both protein fragments were stable and showed polymer ase activity, albeit specific activity and thermostability of the vari ant Delta 413 were significantly decreased compared with the full leng th Taq polymerase, In order to increase the thermostability of the var iant Delta 413, a three-dimensional model of the polymerase domain of Tag polymerase was built by homology with a model of the Klenow fragme nt of the E.coli pol I based on the available C alpha coordinates. Con sequently two variants were designed and constructed using site-direct ed mutagenesis. The strategies used were deletion of 10 flexible amino acids and replacement of two hydrophobic amino acids on the surface b y more hydrophilic ones, Compared with the initial protein fragment, b oth variant enzymes showed an increase in polymerase activity and ther mostability. After the completion of this work, X-ray coordinates of t he Taq polymerase became available from the protein structure data ban k, A comparison between the homology model and the experimental three- dimensional structure proved the quality of the model.