Molecular cloning of a gene possessing streptokinase activity

Abstract

The purified genomic DNA from Streptococcus equisimilis was digested partially with PstI and eluted by electrophoresis to ten fractions according to the size. Fractions 3, 4, 5 and 6 corresponding 7.4kb, 6.7kb, 4.4kb and 3.5kb were ligated with a vehicle, pBR322 which was digested with PstI and dephosphorylated with BAP. The ligated DNA was transformed into competent E. coli HB101 cells and selected transformants showing streptokinase activity by the method of the caseinolytic activity of activated plasminogen. Around 15,000 colonies were appeared on the selection media having tetracycline indicating the colonies are transformants. These transformed colonies were tooth-picked and plates with developed colonies were overlayed with 9m1 of 50mM Tris.Hcl, PH8.1/150mM Nacl, containing 90mg agar, 100g of human plasminogen, and 2ml of Skim milk. Among the 15,000 transformants tested 26 colonies showed clear halo indicating the transformants contain streptokinase gene. The insert size of the plasmid carrying the streptokinase gene was a 2.5kb, 4.3kb and 5.8kb respectively and designated as pDC2.5, pDC3.4 and pDC5.8. The 2.5kb fragment of pDC2.5 was a part of pDC4.3, pDC5.8 hybrid plasmids. The restriction maps of all 3 hybrid plasmids were constructed by digesting with PstI, PvuII, SalI, HindIII, AvaI, BamHI, EcoRI, ClaI, respectively. It was found that E. coli HB101 colonies transformed with three hybrid plasmid produced caseinolysis when tested in any state of growth. Quantitatively, the smaller the insert size, the much product streptokinase was produced. By means of purifying the proteins, I performed the polyacryl-amide gel electrophoresis, $\varepsilon$-ACP ($\varepsilon$-amino caproic acid) inhibition test and soybean trypsin inhibitor inhibition test and it was thought that the cloned gene was streptokinase gene.