Identification of a cDNA clone for bovine tissue factor

Tissue factor is a membrane-bound glycoprotein of approx.43 Kd that is necessary for activation of the extrinsic pathway of blood coagulation. The amino terminal amino acid sequence of purified bovine tissues factor was used to design a synthetic 17-mer oligodeoxyribonucleotide probe. A lambda gtll bovine brain cortex cDNA expression library was screened using both the /sup 32/P-labeled oligodeoxynucleotide probe and polyclonal antibodies to bovine tissue factor. A recombinant phage was isolated which gave a positive reaction with both probes. Cells containing the lambda gtll phage clone produce the tissue factor fragment as a fusion protein with ..beta..-galactosidase. The isolated DNA fragment coding for part of the bovine tissue factor gene is estimated to be approximately 500 bases in length by agarose gel electrophoresis. The ..beta..-glactosidase - tissue factor fusion protein, subjected to Western immunoblotting, shows a protein product of approximately 130 Kd which is reactive to anti-tissue factor antibodies. This corresponds well to the 114 Kd ..beta..-galactosidase plus the predicted approx. 16 Kd fragment of tissue factor. Experiments are in progress to transfer the presumed tissue factor gene fragment into phage M13 for nucleotide sequence analysis.