E. coli에서 사람 프리트롬빈 2 cDNA의 크로닝과 발현

Abstract

A 0.6 kbp of 3'-portion of human prothrombin cDNA was isolated from a liver cDNA library of ${\lambda}gt$ 10 by in situ plaque hybridization. A cDNA fragment corresponding to the N-terminal region of prethrombin 2 was obtained from another cDNA library synthesized by reverse transcription of human liver mRNA using a specific oligonucleotide as a primer. The 48 bp cDNA sequences starting from the first nucleotide to the AvaI site of human prethrombin 2 cDNA was chemically synthesized such that prethrombin 2 can be expressed directly in E. coli expression vector system. The open reading frame(ORF) for human prethrombin 2 was cloned in pUC18 by combining the synthetic fragment and the cDNA fragments. Expression plasmids were constructed in which the ORF for human prethrombin 2 was inserted into the site downstream from the thermoinducible leftward promoter $(P_L)$ and the cII ribosome binding site of bacteriophage ${\lambda}$. Upon temperature induction, the plasmids expressed 34,000 dalton polypeptide, which was accumulated to an amount corresponding to 5-10% of the total bacterial proteins. The 34,000 dalton polypeptide was identified as human prethrombin 2 by SDS-gel electrophoresis and Western blot analysis.