Expression of streptokinase gene under the control of bacteriophage lambda $P_L$ promoter in escherichia coli

Abstract

Streptokinase is an extracellular secretory protein produced by many strains of hemolytic streptococci and shows the activity of lysis of blood clots in the presence of plasminogen. For the production and easy preparation of streptokinase, Roh, D.C. et al. (Korean Biochem. J. (1986) in press) have cloned the streptokinase coding gene skc from the chromosomal DNA of Streptococcus equisimilis ATCC 9542 by shot-gun cloning method with PstI in Escherichia coli. In case that the skc was cloned onto a multicopy plasmid, pBR322, in E. coli, the expression of streptokinase was increased only 2.5 folds comparing with the original strain, S. equisimilis. In this research, in order to overproduce the streptokinase, the previously cloned skc was subcloned to plasmid pPLc2833 which contained the bacteriophage lambda PL promoter. Because the strength of PL promoter is very high and the transcription directed from PL promoter can be easily controlled by the temperature sensitive mutant repressor (cI857), the plasmid pPLc2833 which contained PL promoter was used as a expression vector. In order to construct the streptokinase overproducing plasmid, the skc and pPLc2833 was electroeluted from pSK2.5-I and pPLc2833 after PstI digestion. Because pPLc2833 has two PstI cleavage site, it was partially digested with PstI and electroeluted the single cutted 2.8 kb fragments. After the bacterial alkaline phosphatase treatment to prevent the recircularization of pPLc2833, the single cutted pPLc2833 was ligated with the skc fragment. The ligation mixture was transformed to E. coli M5248 by the method of Hanahan, D. (51) and the colonies which showed the streptokinase activity was screened by the method of casein-plasminogen overlay test (17). Among the 3000 transformants, the 30 colonies could produce the clear zone in the presence of plasminogen. From the restriction enzyme (PstI, HindIII and BamHI) digestion pattern, it was found that all recombinant plasmids contained the skc fragmen...