A Bacillus subtilis endo-β-1,4-glucanse gene, previously cloned in our laboratory, was subcloned in expression vectors, pPLc2833 and pKK223-3. Plasmid pPLc2833 contains lambda $P_L$ promoter followed by multiple cloning site. Plasmid pKK223-3 contains the hybrid promoter tac which has the -35 region of trp promoter and -10 and operator region of lac operon followed by multiple cloning sites originated by pUC8. The endoglucanase gene-containing plasmid, pUBS102(8), was digested with appopriate restriction enzymes, and ligated with the restriction enzymetreated vectors. Those ligation mixtures were used to transform E. coli strains of M5248 and JM103. From the transformants, recombinant plasmids were isolated and named pLBS1 and pKBS1. In pLBS1 the structural gene of the endoglucanase was inserted under the control of promoter $P_L$ from pPLc2833, whereas in pKBS1 the same structural gene was inserted under the control of promoter Ptac from pKK223-3.
E. coli M5248 was transformed with pLBS1 and JM103 was with pKBS1. E. coli M5248(pLBS1) was grown in LB medium at 28℃ until the O.D.600 value reached 0.4-0.5. When the culture temperature was shifted to 42℃ the endoglucanase production was induced to reach up to about 1.5 times of that of uninduced cells in 3 h, and followed by a plateau.
When the culture of E. coli JM103 (pKBS1) was induced with IPTG, the enzyme production reached 15 times higher than that of the uninduced cells. The actual production of the endoglucanase protein was much higher than the enzyme activity and reached 23% of the total cell protein suggesting that the new protein produced was remained in E. coli cells in an inactive form.
본 실험실에서 분리된, 고초균의 섬유소 분해효소 유전자를 발현용 벡터인 pPLc2833 과 pKK223-3에 서브클론 하였다. 이들은 강력한 프로모터인 람다 $P_L$ 과 tac 을 각각 가지고 있는 벡터들이다. 섬유소 분해효소가 제대로 서브클론된 플라스미드들을 각각 pLBS1 과 pKBS1 이라고 명명하였다. 이들을 가지는 대장균 전환주를 가지고, 온도 변화와 IPTG 로 유도되는 효소 생산 실험을 수행하였다.
pLBS1 을 가지는 대장균 전환주는, 온도 변화로 효소 생산을 유도하면 서브클론 하기 전보다 1.5배 정도의 효소 활성을 나타내었다. pKBS1 을 가지는 전환주는 IPTG 로 효소합성을 유도하면, 전체 세포 단백질의 23% 에 해당하는 양의 섬유소 분해효소를 합성하는 것으로 밝혀졌는데, 효소 활성은 서브클론 하기 전의 5배 정도만 증가하였다. 이로부터 생각해 보면, pKBS1 을 가지는 전환주는, IPTG 유도시, 함유체를 형성하는것 같다.
