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 fragment at downstream of the PL promoter and the orientation of transcription of the inserted skc was same or opposite comparing with the orientation of PL promoter. The recombinant plasmid which contained the skc in the same orientation with PL promoter was named as pPS10 and the opposite orientation named as pPS11.
In order to check the stability of the recombinant plasmid in E. coli M5248, the host cells containing pPS10 or pPS11 were grown to saturation in LB medium at 28℃ in the absence of antibiotics and then the plating efficiencies of the cells were checked at 28℃ and 42℃. The recombinant plasmid was considerably stable in E. coli M5248 at 28℃, but very unstable at 42℃. So the expression of the skc was conducted by two stage culture, first the growth stage at 28℃ and the expression stage at 42℃ by derepression of PL promoter resulting from the inactivation of cI857 gene products.
On LB agar plate, the expression level of the skc after 4 hours of temperature induction was not markedly increased in both cases of pPS10 and pPS11 when its activity was examined by the casein/plasminogen overlay test.
In liquid culture, the host cells containing pPS10 or pPS11 showed the sluggish growth comparing with the host cells itself and its growth was ceased after 1-2 hr of the temperature shift to 42℃. In case of the host cells containing pPS10, the expression level of streptokinase was increased about 4 folds comparing with E. coli HB101 containing pSK2.5-I after 6 hr of temperature induction. And in case of pPS11, the expression level of streptokinase was somewhat decreased after the temperature induction.
For more high level expression of streptokinase, it is probably required that the intervening sequence between PL promoter and streptokinase structural gene should be cutted out and the ribosome binding site for translation of streptokinase also should be changed from its native one to E. coli's or bacteriophage's one.
혈전증 치료제로 널리 이용되는 streptokinase를 대장균내에서 대량생산하기 위하여 Streptococcus equisimilis ATCC 9542로 부터 streptokinase coding gene을 pBR322에 cloning하고 이를 대장균내에서 발현시킨 바 있다 (19). 이때 streptokinase의 발현정도는 본래의 균주 보다 수 배 정도 증가하는 것이 관찰되었다. 이 연구의 목적은 streptokinase를 대장균내에서 대량 발현하기 의한 재조합 플라스미드의 제조와 그 최적 발현 조건을 확립하는데 있다.
본 실험에서는 streptokinase의 대량발현을 위하여 streptokinase coding gene (skc)을 PL promoter를 가지고 있는 pPLc2833에 subcloning하고, 이에 의한 transcription 을 temperature sensitive mutant repressor를 만드는 cI857 gene에 의하여 조절하였다. 즉 28℃ 에서는 repressor가 active하게 되어 PL promoter가 repression되는데 비하여, 42℃ 에서는 repressor가 inactive하게되어 PL promoter가 derepression된다.
pPLc2833에는 multiple cloning site내와 β-lactamase gene내 등 두 곳에 PstI 절단부위가 있기 때문에, PstI으로 부분 절단하여 한곳만 절단된 2.8 kb fragment를 electroelution하였다. 이것에 bacterial alkaline phosphatase를 처리하여 recircularization을 방지한후, pSK2.5-I으로부터 PstI을 써서 잘라낸 skc를 포함하는 2.5 kb fragment와 ligation하였다. 이 ligation mixture를 high efficient Hanahan method에 의하여 E. coli M5248에 transformation한 후 casein/plasminogen overlay test에 의하여 plasminogen 존재시 casein hydrolysis 능력이 있는 30개의 colony를 분리하였다. 이들로 부터 plasmid를 분리하여 제한효소로 잘라본 결과 skc가 pPLc2833내의 PL promoter와 transcription 방향이 같은 것과 반대인 것이 모두 발견되었으며, 이들을 각각 pPS10과 pPS11이라 명명하였다.
이 재조합 plasmid는 28℃로 culture할 경우 E. coli M5248내에서 상당히 안정하나 42℃ 에서는 매우 불안정하다. 따라서 streptokinase의 대량발현은 먼저 28℃에서 cell을 키운 후 culture temperature를 42℃로 올려서 PL promoter를 derepression 시키는 방법을 이용했다.
LB agar plate 상에서 실험할 때 casein/plasminogen overlay test에 의하여 보면 temperature induction에 의한 streptokinase의 발현증가가 크지 않음을 알 수 있었다. 액체배양을 하면 pPS10의 경우 6 시간 temperature induction에 의하여 원래의 pSK2.5-I 보다 약 4배 정도의 발현증가가 관찰되었다. 따라서, 좀 더 대량발현을 위해서는 streptokinase의 structural gene을 PL promoter에 좀 더 가까이 연결시키고, 또한 streptokinase의 translation을 위한 ribosome- binding site도 현재의 streptococcal ribosome-binding site에서 대장균이나 bacteriophage의 그것으로 바꾸는 것이 필요하다고 생각된다.
