Cloning of full-length cDNA coding for human prothrombin and its expression in monkey COS cells

Abstract

cDNA fragments coding for the leader sequence and N-terminal region of human prothrombin was isolated from partical cDNA clones that were enriched for prothrombin cDNA clones. Poly (A)+RNA was isolated from human liver and size-fractionated by sucrose gradient centrifugation. Poly (A)+RNAs in the ranges of 1500 to 4000 nucleotides were pooled and utilized for cDNA synthesis by reverse transcription using a specific oligodeoxynucleotide as a primer. Among about 2000 colonies, eight positive cDNA clomes were obtained by hybridization with ³²P-labeled oligodeoxynucleotide complementlary to 5'-termonal region of prothrombin mRNA sequences. Six of the recombinant plasmids contained prothrombin cDNA inserts of expected size. Restriction and sequencing analysis showed that the cDNA insert of phII3 plasmid contained 12 nucleotides of 5'-untranslated region and the DNA sequences conding for the leader and N-terminal 312 residues of human prothrombin. The complete cDNA sequence coding for the leader and 579 amono acid residues of human prothrombin was cloned in pUC18 by combining the two overlapping cDNA inserts of phII3 amd pHPT2 (Choi et al., 1989). The full-lenth prothromibin cDNA was inserted into the multiclonung sites downstream from the SV40 late promoter of pSLS plasmid. Upon transfection with the recombinant lpasmid, human prothrombin was expressed in SV40-tranformed African green monkey kidney(COS) cells, and was exclusively found in the growth medium.