대장균에서 스트렙토키나제 유전자의 대량발현 및 aprE-lacUV5 융합 전사촉진제의 개발

학위논문(박사) - 한국과학기술원 : 생물공학과, 1991.2, [ xi, 160 p. ]The expression vector for streptokinase has been constructed from our previously cloned streptokinase coding gene(\underline{\mbox{skc}}). Because of its deleterious effect on the host cell growth, the leader sequence of skc was removed and the leader sequence-deleted \underline{\mbox{skc}} was subcloned into the vector pkk223-3, which contains the regulatable \underline{\mbox{tac}} promoter and \underline{\mbox{rrnB}} $T_1T_2$ transcription terminator, with a short synthetic oligonucleotide adapter. When this vector, pKS601, was expressed in \underline{\mbox{Escherichia}} \underline{\mbox{coli}}, a 47.4 kD protein was found to be newly accumulated to about 12% of the total cellular proteins and it was identified as the streptokinase by immunoblotting with rabbit anti-streptokinase polyclonal serum. The expressed leader sequence-deleted streptokinase caused no detrimental effect to host cell physiology, which means that the deleterious effect of the wild-type \underline{\mbox{skc}} had caused by its putative leader sequence, and showed no degraded 44 kD streptokinase, which is characteristic feature of streptokinase expressed from wild-type \underline{\mbox{skc}}. At 37℃, one-fourth of streptokinase was found in sonication-insoluble pellet, which regarded as inclusion bodies, but at 25℃, nearly all of streptokinase was found in sonication-soluble fraction. But the total activity of expressed streptokinase showed the highest values when cells had cultured and induced at 37℃, The expressed streptokinase was purified to near homogeneity using DEAE-cellulose and Sephadex G-150 column. Its specific activity was 1.3×$10^5$ CLN units/mg protein. And a series of \underline{\mbox{E.}} \underline{\mbox{coli}}-\underline{\mbox{Bacillus}} \underline{\mbox{subtilis}} shuttle vectors was constructed by fusing and manipulating the \underline{\mbox{Staphylococcus}} \underline{\mbox{aureus}} plasmid pUB110 and \underline{\mbox{E.}} $\underline{\m...한국과학기술원 : 생물공학과

대장균에서 bacteriophage lambda 의 PLP_L promotor 에 의한 streptokinase 유전자의 발현

학위논문(석사) - 한국과학기술원 : 생물공학과, 1987.2, [ x, 54 p. ]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...한국과학기술원 : 생물공학과