CAJ | 학술논문

目的确定在一个质粒载体上串联目的操纵子以提高目的蛋白表达量的可行性,阐明宿主细胞对胞内质粒DNA总量调控的可能机制.方法亚克隆构建了两组操纵子正向串联的表达质粒:CWll系列分别含1-4个正向操纵子,质粒大小以2.25 kb的增加量从5.47 kb增加至12.26 kb;CW12系列分别含1-3个正向操纵子,质粒大小以2.16 kb的增加量从5.40 kb增加至9.72 kb.SDS凝胶电泳和激光密度扫描测定目的蛋白表达量;3H-TdR掺入法测定质粒拷贝数.结果操纵子的串联不影响宿主大肠杆菌的生长;温度诱导表达后CW11系列目的蛋白表达量分别为菌体总蛋白的44.9%±3.9%、51.3%±4.1%、54.8%±3.3%和58.2%±3.4%,CW12系列目的蛋白表达量分别为菌体总蛋白的32.2%±5.0%、42.8%±4.1%和46.9%±4.0%.两组质粒的拷贝数均随操纵子串联个数的增加而显著减少(P<0.01),但目的基因的总剂量随之显著增加(P<0.01),而同一系列的质粒在每个宿主细胞内的质粒DNA总量没有显著的变化(P>0.05).结论操纵子串联增加了目的基因的剂量从而提高了目的基因在大肠杆菌中的表达水平.质粒大小和其拷贝数呈负相关,在同一培养条件下,对于特定的大肠杆菌菌株,同一系列的质粒在宿主细胞内的DNA总量在一定程度上相对恒定.
목적학정재일개질립재체상천련목적조종자이제고목적단백표체량적가행성,천명숙주세포대포내질립DNA총량조공적가능궤제.방법아극륭구건료량조조종자정향천련적표체질립:CWll계렬분별함1-4개정향조종자,질립대소이2.25 kb적증가량종5.47 kb증가지12.26 kb;CW12계렬분별함1-3개정향조종자,질립대소이2.16 kb적증가량종5.40 kb증가지9.72 kb.SDS응효전영화격광밀도소묘측정목적단백표체량;3H-TdR참입법측정질립고패수.결과조종자적천련불영향숙주대장간균적생장;온도유도표체후CW11계렬목적단백표체량분별위균체총단백적44.9%±3.9%、51.3%±4.1%、54.8%±3.3%화58.2%±3.4%,CW12계렬목적단백표체량분별위균체총단백적32.2%±5.0%、42.8%±4.1%화46.9%±4.0%.량조질립적고패수균수조종자천련개수적증가이현저감소(P<0.01),단목적기인적총제량수지현저증가(P<0.01),이동일계렬적질립재매개숙주세포내적질립DNA총량몰유현저적변화(P>0.05).결론조종자천련증가료목적기인적제량종이제고료목적기인재대장간균중적표체수평.질립대소화기고패수정부상관,재동일배양조건하,대우특정적대장간균균주,동일계렬적질립재숙주세포내적DNA총량재일정정도상상대항정.
Objective To examine the feasibility of linking operons in tandem to enhance expression of heterologous genes in Escherichia coli (E. coli) and clarify the potential control mechanism of the total plasmid DNA amount in each host cell.Methods Two series of expression plasmids, CW11 and CW12, containing 1 to 4 and 1 to 3 heterologous gene operon(s) respectively, were constructed. The molecular size of the CW11 series varied from 5.47 kb to 12.26 kb in 2.25 kb increments. The CW12 series varied from 5.40 kb to 9.72 kb in 2.16 kb increments. The expression level of desired protein was assayed by SDS-PAGE and laser density scanning. Plasmid copy number was determined by incorporation with 3 H-thymidine (3H-TdR). Results No influence of the tandem-joined operons on host growth and plasmid stability was observed. Upon induction, the desired protein accumulations in the CW11 series were 44.9%±3.9%, 51.3%±4.1%, 54.8%±3.3% and 58.2%±3.4% of total cell protein. In the CW12 series, the yields were 32.2%±5.0%, 42.8%±4.1% and 46.9%±4.0% of total cell protein. As size increased, the plasmid copy number decreased, but target gene dosage increased significantly (P<0.01). Further calculation showed that the total amount of plasmid DNA per cell was not significantly different in each series (P>0.05) and restricted to some extent. Conclusions Increasing the target gene dosage by tandem linking of operons may enhance the expression level of a desired protein. Although the size (kb) and the copy number of each plasmid are negatively interrelated, for certain plasmids in each series, their total DNA amount per cell seems to be a restricted constant for specific E. coli strains under identical incubation condition.