원문정보
Plasmid Propagation and Heterologous Gene Expression in Recombinant Yeast
초록
영어
The effects of genetic and environmental factors on productivity of a cloned protein were studied in recombinant Saccharomyces cerevisiae. Plasmid stability and copy level were very high for a REP+ system(at ca. 10 generations, stability: 65-90%, plasmid copy number per cell: 40-200), whereas these were very low for a yep- system(at ca. 10 generations, stability: 30%, plasmid copy number per cell 20). In plasmids containing the 2um circle genome, a[ciro] strain was a preferred host cell since the plasmid stability and the copy number in a [ciro] strain were higher than in a [cir+]strain. Cloned gene expression was dependent on plasmid copy number and stability. The inducer (galactose) level played a very important role in cloned lacZ gene expression, showing that a galactose concentration of 0.8% was sufficient for induction of gene expression. Induction rate was very fast in the case of plasmids exhibiting high stability and copy number by a factor of 4 to 25. The time to reach the peak value of gene expression was longer when galactose was added at the start of fermentation (ca. 26 hours) than at the mid-exponential phase (ca. 6 hours). Glucose repression was reduced by a factor of 2 to 5 as the relative inducer level increased.
한국어
효모균에서의 유전자 재조합에 의한 단백절 생산에 미치는 유전학적, 환경적인 요인의 영향을 연구하였. Plasmid 안정도와 개수는 REP+ 체계 에서 대단히 높은 반면, rep 체계에서는 매우 낮았다. 2um circle plasmid genome을 포함하는 plasmid의 경우에 있어서. [ciro] 형 세포에서의 plasmid 안정도와 개수가 [cir+]형 세포에서보다 높기때문에 [cir+o]형 세포가 더 선호되는 세포였다. 유전자 발현은 plasmid 개수와 안정도에 좌우 되었다. 촉진제의 양이 유전자 발현에 매우 중요 한 역할을 했다. 유전자 발현의 촉진에 필요한 g떠actose의 농도는 0.8% 이 변 충분했다. 높은 안정 도와 개수를 갖는 plasmid의 경우 촉진속도는 매우 빨랐다. Galactose가 배양의 시작부분부터 첨가 될 때가 mid-exponential ph잃e에 첨가될 때보다 유전자 발현의 극대점에 이르는 시간이 걸었다. 상대적 촉진제의 양이 증가함에 따라 glucc잉e억제 현상은 감소되었다
목차
INTRODUCTION
MATERIALS AND METHODS
Strains and Plasmids
Media
Culture Conditions
General Procedures
RESULTS AND DISCUSSION
Host-Vector Interactions
Cloned Gene Expression
Batch Induction Behavior
요약
REFERENCES